Vincent Praloran
email: vincent.praloran@u-bordeaux2.fr

Keywords: hematopoiesis, hypoxia, cell cycle, quiescence, differentiation, progenitors, p27, FDCP-Mix O 2 , Oxygen, HIF-1, hypoxia-inducible factor-1, Epo, Erythropoietin, VEGF, Vascular Endothelial Growth Factor, HSC, Hematopoietic stem cell, PI, Propidium Iodide, CFSE, carboxyfluorescein diacetate succinimidyl diester, MFI, Mean Fluorescence Intensity Repopulating Ability, CFC, Colony Forming Cell, LC, Liquid Culture, KIP, Kinase Inhibitor Protein, CDK, Cyclin Dependant Kinase

Oxygen concentrations in bone marrow vary from 4% in capillaries to less than 0.1% in subendosteum where hematopoietic stem cells reside in specific niches. Culture at low O 2 concentrations (3, 1 and 0.1%) influences hematopoietic stem and progenitor cells survival, proliferation and differentiation, depending on their level of differentiation. Culture of human CD34 + cells at low O 2 concentrations (O 2 ≤ 3%) maintains stem cell engraftment potential better than at 20% O 2 (NOD/Scid xenograft model). In contrast, progenitors disappear from cultures at/or below 1% O 2 concentrations. A very low O2 concentration (0.1%) induces CD34+ quiescence in G 0 . The exploration of molecules and mechanisms involved in hematopoietic stem and progenitor cells' quiescence and differentiation related to low O2 concentrations is unfeasible with primary CD34 + cells. Therefore, we performed it using murine hematopoietic non leukemic FDCP-Mix progenitor cell line. The culture of the FDCP-Mix line at 0.1% O 2 induced in parallel G 0 quiescence and granulo-monocytic differentiation of most cells, while a minority of undifferentiated self-renewing cells remains in active cell cycle. Hypoxia also induced hypophosphorylation of pRb and increased the expression of p27 KIP1 , two proteins that play a major role in the control of G 0 and G 1 to S phase transition.

Introduction

Physiological oxygen (O 2 ) concentrations in tissues are much lower than in atmospheric air (20%), ranging from 14% in pulmonary alveoli to less than 1% in several organs [START_REF] Ivanovic | Hypoxia or in situ normoxia: The stem cell paradigm[END_REF].

Numerous publications demonstrate the major role of variations of O 2 concentration in the responses of normal cells, transformed cells and leukemic cells from primoculture or from continuous cell lines [START_REF] Desplat | Hypoxia modifies proliferation and differentiation of CD34(+) CML cells[END_REF][START_REF] Giaccia | The biology of hypoxia: the role of oxygen sensing in development, normal function, and disease[END_REF][START_REF] Giuntoli | Severe hypoxia defines heterogeneity and selects highly immature progenitors within clonal erythroleukemia cells[END_REF][START_REF] Ivanovic | Simultaneous Maintenance of Human Cord Blood SCID-Repopulating Cells and Expansion of Committed Progenitors at Low O2 Concentration (3%)[END_REF][START_REF] Nguyen-Khac | Functional analyses of the TEL-ARNT fusion protein underscores a role for oxygen tension in hematopoietic cellular differentiation[END_REF][START_REF] Schmaltz | Regulation of proliferationsurvival decisions during tumor cell hypoxia[END_REF][START_REF] Giuntoli | Hypoxia suppresses BCR/Abl and selects imatinib-insensitive progenitors within clonal CML populations[END_REF]. Low O 2 concentration induces apoptosis or cell survival, proliferation or quiescence, self renewal or differentiation depending on the degree of hypoxia, the cell type and the level of differentiation.

Response to hypoxia is largely dependent of hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor. At O 2 concentrations lower than 5%, the HIF-1α subunit is no longer degraded by the proteasome [START_REF] Berra | HIF prolylhydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1alpha in normoxia[END_REF][START_REF] Jiang | Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension[END_REF] and dimerizes with HIF-1β. The active complex then induces the expression of numerous genes including those of growth factors (Erythropoietin (Epo) and Vascular Endothelial Growth Factor (VEGF)), transcription factors and cell cycle proteins [START_REF] Hackenbeck | HIF-1 or HIF-2 induction is sufficient to achieve cell cycle arrest in NIH3T3 mouse fibroblasts independent from hypoxia[END_REF][START_REF] Ivanovic | Physiological, ex vivo cell oxygenation is necessary for a true insight into cytokine biology[END_REF][START_REF] Mole | Genome-wide Association of Hypoxia-inducible Factor (HIF)-1{alpha} and HIF-2{alpha} DNA Binding with Expression Profiling of Hypoxia-inducible Transcripts[END_REF][START_REF] Vengellur | Gene expression profiling of hypoxia signaling in human hepatocellular carcinoma cells[END_REF].

In bone marrow, hematopoietic cells are permanently exposed to O 2 concentrations ranging from 4% in capillaries to less than 0.1% in endosteal areas [START_REF] Chow | Modeling pO(2) distributions in the bone marrow hematopoietic compartment. II. Modified Kroghian models[END_REF][START_REF] Chow | Modeling pO(2) distributions in the bone marrow hematopoietic compartment. I. Krogh's model[END_REF][START_REF] Tondevold | Observations on long bone medullary pressures in relation to arterial PO2, PCO2 and pH in the anaesthetized dog[END_REF] where the most primitive stem cells reside [START_REF] Nilsson | Spatial localization of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches[END_REF][START_REF] Parmar | Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia[END_REF]. Hematopoietic stem cells (HSC) are better preserved in cultures at low O 2 (1 to 3 %) than at atmospheric O2 concentration (20%) [START_REF] Ivanovic | Simultaneous Maintenance of Human Cord Blood SCID-Repopulating Cells and Expansion of Committed Progenitors at Low O2 Concentration (3%)[END_REF][START_REF] Danet | Expansion of human SCIDrepopulating cells under hypoxic conditions[END_REF][START_REF] Ivanovic | Primitive human HPCs are better maintained and expanded in vitro at 1 percent oxygen than at 20 percent[END_REF]. We previously showed that culture at 0.1% O 2 prevents cord blood G 0 CD34 + cells entry in active cell cycle and induces those in active proliferation to re-enter in G 0 [START_REF] Hermitte | Very low O2 concentration (0.1%) favors G0 return of dividing CD34+ cells[END_REF]. The effects of low O 2 concentration on the survival, proliferation and differentiation of normal and leukemic hematopoietic progenitors are more controversial. Indeed, in some articles a prodifferentiative effect [START_REF] Nguyen-Khac | Functional analyses of the TEL-ARNT fusion protein underscores a role for oxygen tension in hematopoietic cellular differentiation[END_REF][START_REF] Huang | Cobalt chloride and low oxygen tension trigger differentiation of acute myeloid leukemic cells: possible mediation of hypoxia-inducible factor-1alpha[END_REF][START_REF] Song | Hypoxia-inducible factor-1alpha-induced differentiation of myeloid leukemic cells is its transcriptional activity independent[END_REF][START_REF] Zhang | Hypoxia-HIF-1alpha-C/EBPalpha/Runx1 signaling in leukemic cell differentiation[END_REF] was pointed out while in others it was suggested that oxygen levels below 1% block cell proliferation and induce their rapid disappearance from cultures, probably by inducing apoptosis [START_REF] Giuntoli | Severe hypoxia defines heterogeneity and selects highly immature progenitors within clonal erythroleukemia cells[END_REF][START_REF] Ivanovic | Primitive human HPCs are better maintained and expanded in vitro at 1 percent oxygen than at 20 percent[END_REF][START_REF] Kovacevic-Filipovic | Interleukin-6 (IL-6) and low O(2) concentration (1%) synergize to improve the maintenance of hematopoietic stem cells (pre-CFC)[END_REF][START_REF] Ivanovic | Hypoxia maintains and interleukin-3 reduces the pre-colony-forming cell potential of dividing CD34(+) murine bone marrow cells[END_REF].

Culture at low O 2 concentration (0.5%) causes cell cycle arrest in G 1 of mouse embryonic fibroblasts and splenic B lymphocytes by inducing Rb hypophosphorylation and p27 KIP1 upregulation [START_REF] Goda | Hypoxiainducible factor 1alpha is essential for cell cycle arrest during hypoxia[END_REF][START_REF] Gardner | Hypoxia inhibits G1/S transition through regulation of p27 expression[END_REF]. All these results led us to explore the effects of low O 2 concentration on the proliferation and differentiation of hematopoietic progenitors. Extensive protein studies using normal human or murine primoculture progenitor cells were quite unfeasible for two reasons: heterogeneity of the CD34 + cell population and difficulty of obtaining them in large numbers. Since the murine cell line FDCP-Mix, which is less heterogeneous, has major features of normal hematopoietic progenitors, we tested its response to hypoxia. Indeed, FDCP-mix cells have a normal karyotype, are non-leukemic, strictly depend on IL-3 for their survival and proliferation, and differentiate towards different myeloid lineages when cultured with other cytokines [START_REF] Heyworth | The role of hemopoietic growth factors in self-renewal and differentiation of IL-3-dependent multipotential stem cells[END_REF]. Varying the concentrations of O 2 from 20 to 0.1% in cultures of FDCP-mix cells had a time -dependent effect on their proliferation and cell cycle, with cells reaching quiescence (G 0 ) after 72h of culture at 0.1% O 2 . Then we used these cells to analyze the self renewal/proliferation/differentiation balance and the variations of p27 KIP1 levels and pRb phosphorylation, two proteins potentially involved in G 0 cell cycle arrest.

Results

Low O 2 concentration inhibits FDCP-Mix cell growth

Low O 2 concentration induced a time-dependent FDCP-Mix growth inhibition (fig 1a). The number of living cells at 24h was not significantly different in 20% O 2 vs. 0.1% O 2 concentration cultures, neither was their doubling time (about 30h in both conditions) (fig 1b).

At later time points, the growth of FDCP-Mix cells cultured at 0.1% O 2 was dramatically reduced, with a doubling time that reached 104h after more than 48h of culture. Since the percentage of apoptotic cells in two conditions was similar (fig 1c) after 24h, 48h and 72h of culture, we concluded that the growth arrest observed at 0.1% O 2 was due to the inhibition of proliferation of FDCP-Mix cells after 24h.

Very low O 2 concentration (0.1%) induces FDCP-Mix cell arrest in G 0

We then analyzed the FDCP-Mix cell cycle status after 24, 48 and 72h of culture at 20 and 0.1% O 2, using double labeling with Propidium Iodide (PI, a quantitative DNA fluorescent dye) and anti Ki-67 antibodies (Ki-67 antigen is expressed in the nucleus of cycling cells and absent from G 0 cells) to discriminate cells in G 0 (DNA =2N; Ki-67 -) from cells in G 1 (DNA =2N; Ki-67 + ), S (2N< DNA <4N; Ki-67 + ) and G 2 /M (DNA =4N; Ki-67 + ). As shown in fig 2a, the percentage of FDCP-Mix cells in G 0 remained stable (13.6±2.3%) during the 72h of culture at 20% O 2 . In contrast, at 0.1% O 2 the percentage of G 0 cells significantly increased to 48.8±5.4% after 72h of culture, while the percentage of cells in G 1 , S and G 2 M decreased in

Very low O 2 concentration (0.1%) induces the differentiation of a majority of FDCP-

Mix cells

May-Grünwald-Giemsa staining of FDCP-Mix cytospins showed that after 72h of culture at 0.1% O 2 , the majority of cells acquired a granulo-monocytic differentiation (fig 4a) that was confirmed by FCM analysis of the expression of Gr-1 (granulocytic differentiation marker), Mac-1 (monocytic differentiation marker), as well as CD34 and CD117 (markers of primitive hematopoietic progenitors) cell surface markers (fig 4b, c andd). Indeed, after 3 days of culture at 0.1% O 2, the percentage of cells positive for Gr-1 and CD117 respectively increased from 26 to 63% and decreased from 27 to 14 % (fig 4b andc). In parallel, as expected, we observed a decrease of the Mean Fluorescence Intensity (MFI) of the CD34 primitive marker together with an increase of the MFI of the granulo-monocytic marker Mac-1 and Gr-1 (fig 4d). These results confirmed that culture at 0.1% O 2 induces the granulo-monocytic differentiation of the FDCP-Mix cell line.

Very low O 2 concentration (0.1%) also maintains a minor population of FDCP-Mix cells with culture-repopulating ability (CRA)

FDCP-Mix cells issued from 72h of LC1 at 0.1% O 2 were sequentially replated every 72h at 1.10 5 cells/ml at 20% O 2 to test their ability to repopulate later liquid cultures (LC2 to LC5), as determined by counting the total number of cells in LC2 to LC5 (CRA cell ). The growth rate of cells incubated in LC1 at atmospheric O2 concentration remained stable throughout the experiment (left part of the graph fig 5a). The growth rate of cells issued from LC1 incubated at 0.1% O 2 (right part of the graph fig 5a) in non-selective, normoxic LC2 to LC5 (fig 5a), remained stable during LC2 and LC3, increased during LC4 and reached the growth rate exhibited by cells incubated in normoxic LC1 during LC5. In the meantime the percentage of cells in G 0 decreased and the percentage of cells in G 1 , S and G 2 M started to increase during LC2 to reach control levels at LC4 (data not shown). In order to confirm that this progressive repopulation of LC was due to the maintenance in hypoxia of a limited cell subpopulation, cells rescued from non-selective (normoxic) LC2 to LC5 were cultured in semisolid medium to identify and count, after 11 days, the number of colonies issued from CFC. The percentage of these CRA CFC at the end of 0.1% O 2 LC1 was 14 times lower than after 20% LC1 and progressively returned to similar values in LC4 (fig 5b).

Relationships between G 0 quiescence, Gr-1 expression and CRA CFC cells.

Since the differentiation after 3 days of culture at 0.1% O 2 was accompanied by the G 0 entry of a large percentage of FDCP-Mix cells, we explored the relationship between these two phenomena. As a first step, we sorted Gr-1 + and Gr-1 -cells after 3 days of LC1 at 20 or 0.1% O 2 . The microscopic observation on May-Grünwald-Giemsa cytospins (fig 6a) confirmed that Gr-1 + cells had a typical morphology of terminally differentiated granulocytes including donut-shaped nuclei (arrows ►) whereas Gr-1-cells kept round nuclei with more basophilic cytoplasm as observed in control culture conditions (fig 4a). At both O 2 concentrations all CRA cell -and CRA CFC -type cells were contained within the Gr-1 -population (fig 6b andc).

Analysis of the cell cycle status of Gr-1 + and Gr-1 -cells showed that CRA-type undifferentiated Gr-1 -cells were mostly cycling, while differentiated Gr-1 + cells without CRA were in G 0 (fig 6d). In addition, a 24 hours treatment of FDCP-mix cells with 5FU (10µg/mL) at the end of LC1 followed by medium renewal and replating in LC2, dramatically decreased CRA cell and CRA CFC similarly in both O 2 conditions (data not shown). Altogether, these data demonstrate that at very low 0.1% O 2 concentration, most FDCP-Mix cells differentiate and enter in the quiescent G 0 status while a minority of undifferentiated cells, those which maintain the self renewal of the cell line, remain in active cell cycle.

FDCP-Mix cells in G 0 overexpress hypophosphorylated pRb and p27 KIP1 proteins

Due to their role in the regulation of cell cycling, we analyzed the total amount of p27 KIP1 and pRb proteins and the phosphorylation status of pRb after 24, 48 and 72h of culture at 20% O 2 and 0.1% O 2 . As expected, the total quantity of pRb protein remained stable and similar during the 72h culture at 20 and 0.1% O 2 (fig 7a). S phase transition is dependent on pRb phosphorylation on several specific sites. Therefore, we studied this protein phosphorylation status. Indeed, unphosphorylated pRb progressively increased while phosphorylated pRb disappeared during the 72h culture of FDCP-Mix cells at 0.1% O 2 (data not shown). We then analyzed several pRb phosphorylation sites involved in the G 1 to S phase transition. The ser 608, ser 807/811 and thr 821 sites (stably phosphorylated at 20% O 2 ) became very faint at 0.1% O 2 (fig 7b). In the meantime, the p27 KIP1 level in FDCP-Mix cells strongly increased during cultures at 0.1% O 2 while it remained stable at 20% O 2 (fig 8a). Up-regulation of the p27 KIP1 protein by hypoxia in FDCP-Mix cells was partly related to transcriptional mechanisms since p27 KIP1 mRNA level significantly increased during cultures at 0.1% O 2 (2.76, 3 and 2.4 fold at 24, 48 and 72h respectively) (fig 8b). In order to know if the regulation of the p27 KIP1 protein was linked to the G 0 quiescence and differentiation of the FDCP-Mix cell line, we explored its expression in the Gr-1 -and Gr-1 + populations by flow cytometry. Both the level of p27 KIP1 fluorescence intensity (fig 8c andd) and the percentages of cells expressing high levels of p27 KIP1 were different in Gr-1 -and Gr-1 + cells.

Discussion

In the last ten years, an increasing number of publications were devoted to the physiological low bone marrow O 2 concentrations (from < 0.1% O 2 in sub-endosteal bone marrow niches to 4-5% in vascular areas; compared to 21% in atmospheric air) and to the exploration of their role in the regulation of hematopoiesis [START_REF] Ivanovic | Hypoxia or in situ normoxia: The stem cell paradigm[END_REF][START_REF] Chow | Modeling pO(2) distributions in the bone marrow hematopoietic compartment. II. Modified Kroghian models[END_REF][START_REF] Chow | Modeling pO(2) distributions in the bone marrow hematopoietic compartment. I. Krogh's model[END_REF]. We and others showed that varying O 2 concentrations (from 20 to 0.1%) in cultures of hematopoietic stem and progenitor cells modified their quiescence vs. proliferation and self renewal vs. differentiation balances (2, 4-7, 19-23, 31-33). To investigate the molecules and mechanisms involved in these phenomena we used the FDCP-Mix hematopoietic cell line that is considered to share numerous phenotypic and functional properties with bone marrow hematopoietic progenitors. Indeed, FDCP-mix cells have a normal karyotype, are non-leukemic, strictly depend on IL-3 for their survival and proliferation, and differentiate towards different myeloid lineages when cultured with other cytokines [START_REF] Heyworth | The role of hemopoietic growth factors in self-renewal and differentiation of IL-3-dependent multipotential stem cells[END_REF]. In addition, when cultured at 0.1% O 2 cycling FDCP-Mix cells line progressively accumulated in G 0 after one to few divisions. This accumulation in G 0 was accompanied by a major granulo-monocytic differentiation as shown by sorting Gr-1 + (differentiated) and Gr-1 -(undifferentiated) FDCP-Mix cells after 3 days of culture at 20 and 0.1% O 2 . In both O 2 conditions the Gr-1 -FDCP-Mix subset had a high CRA and a high percentage of cycling cells whereas the Gr-1 + differentiated fraction was mostly in G 0 with a quite null CRA. This result suggests a link between cell cycle arrest and differentiation. Some articles found a differentiating effect of low O 2 concentrations on leukemic cells either from primoculture or from cell lines [START_REF] Nguyen-Khac | Functional analyses of the TEL-ARNT fusion protein underscores a role for oxygen tension in hematopoietic cellular differentiation[END_REF][START_REF] Huang | Cobalt chloride and low oxygen tension trigger differentiation of acute myeloid leukemic cells: possible mediation of hypoxia-inducible factor-1alpha[END_REF][START_REF] Song | Hypoxia-inducible factor-1alpha-induced differentiation of myeloid leukemic cells is its transcriptional activity independent[END_REF][START_REF] Zhang | Hypoxia-HIF-1alpha-C/EBPalpha/Runx1 signaling in leukemic cell differentiation[END_REF] similar to the one observed with FDCP-Mix cells. In contrast, the group of P. Dello Sbarba showed that when the K562 cell line (issued from a blast-crisis chronic myeloid leukemia -an HSC malignancy-patient) was cultured at 0.1% O 2 for 7 days, the number of cells dramatically decreased (by 1 log), most cells underwent apoptosis while a minor hypoxia-resistant subpopulation repopulated LC2 incubated at 20% O 2 , exhibiting full CRA maintenance [START_REF] Giuntoli | Hypoxia suppresses BCR/Abl and selects imatinib-insensitive progenitors within clonal CML populations[END_REF]. Using the murine MEL cell line, they obtained similar results, founding, in addition, at 0.1% O 2 a quiescent subset of CRA cells that resisted to 5FU treatment (4). However, in those two articles, cell differentiation was not studied. When the FDCP-Mix cell line was cultured under similar conditions, apoptosis did not increase significantly, most cells underwent proliferation arrest and differentiated, while a small population of slowly-cycling CRA cells (since killed by 24h of 5-FU) persisted after 3 and even 7 days of culture at 0.1% O 2 . These differences between FDCP-Mix (and the leukemic cell lines that differentiate in hypoxia) on one hand and K562, MEL and CD34 + cells on the other hand could be due 1) to the fact that FDCP-Mix and the leukemic cell lines that differentiate in hypoxia [START_REF] Nguyen-Khac | Functional analyses of the TEL-ARNT fusion protein underscores a role for oxygen tension in hematopoietic cellular differentiation[END_REF][START_REF] Huang | Cobalt chloride and low oxygen tension trigger differentiation of acute myeloid leukemic cells: possible mediation of hypoxia-inducible factor-1alpha[END_REF][START_REF] Song | Hypoxia-inducible factor-1alpha-induced differentiation of myeloid leukemic cells is its transcriptional activity independent[END_REF][START_REF] Zhang | Hypoxia-HIF-1alpha-C/EBPalpha/Runx1 signaling in leukemic cell differentiation[END_REF] derive from progenitors and not from HSC, 2) to their lower sensitivity to apoptosis than primary CD34 + cells in hypoxic cultures owing to their immortalized status (but this does not apply to the K562 and MEL cell lines) or 3) to an association of both phenomena.

Our previous results showing that primoculture progenitors disappear rapidly during cultures of murine bone marrow or human CD34 + cells at 1% O 2 [START_REF] Ivanovic | Primitive human HPCs are better maintained and expanded in vitro at 1 percent oxygen than at 20 percent[END_REF][START_REF] Ivanovic | Hypoxia maintains and interleukin-3 reduces the pre-colony-forming cell potential of dividing CD34(+) murine bone marrow cells[END_REF] while leukemic and FDCP-Mix cell lines survive and differentiate strengthen the hypothesis that the maturation of FDCP-Mix cells in hypoxia is linked to their acquired immortalized status. This allows their prolonged survival as differentiated cells in hypoxia, while primoculture progenitors rapidly die in these conditions before reaching maturation.

The permanent cycling of CRA-type and the absence of apoptosis in cultures of FDCP-Mix cells at very low O 2 concentration that contrasts with the apoptosis of a large percentage and the quiescence of a minority of primoculture CD34 + and MEL cells cannot be explained by the immortalized status of FDCP-Mix since MEL is also a permanent (and leukemic) cell line.

We suggest that this major functional difference in response to very low O 2 concentrations is due to the "stem" or "progenitor" cell status of these different cell types. Together, the abovementioned results with primary CD34 + cells, K562 cells, other leukemic cell lines and our present work imply that regulation by very low O 2 concentrations (<1%) of cycling, differentiation and survival, is depending on the differentiation level of cells (stem cells vs. progenitors) whether normal or leukemic tissue is in question.

These results led us to explore the molecules involved in the cell cycle modifications induced by hypoxia. The G 0 and/or G 1 (undefined since neither Ki-67 nor any other G 0 marker was used) quiescence of fibroblasts and B lymphocytes induced by serum starvation or hypoxia is linked to p27 KIP1 increase that blocks the Cyclin/Cyclin Dependent Kinase (CDK) activity, thus leading to pRb hypophosphorylation [START_REF] Hackenbeck | HIF-1 or HIF-2 induction is sufficient to achieve cell cycle arrest in NIH3T3 mouse fibroblasts independent from hypoxia[END_REF][START_REF] Goda | Hypoxiainducible factor 1alpha is essential for cell cycle arrest during hypoxia[END_REF][START_REF] Gardner | Hypoxia inhibits G1/S transition through regulation of p27 expression[END_REF][START_REF] Besson | A pathway in quiescent cells that controls p27Kip1 stability, subcellular localization, and tumor suppression[END_REF][START_REF] Coats | Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle[END_REF][START_REF] Rivard | Abrogation of p27Kip1 by cDNA antisense suppresses quiescence (G0 state) in fibroblasts[END_REF][START_REF] Kops | Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors[END_REF]. Conversely phosphorylations of pRb on multiple ser/thr sites by Cyclin/CDK during the G 1 phase lead to the release of molecules required for the transition to S phase [START_REF] Adams | Regulation of the retinoblastoma tumor suppressor protein by cyclin/cdks[END_REF]. Briefly, the phosphorylation of thr 821 releases histone deacetylase; the phosphorylation of ser 807/811 releases c-abl and the phosphorylation of ser 608 participates in the release of the master transcription factor E2F.

These three molecules play a major role in the G 1 to S transition. Hypoxia dramatically reduced the phosphorylation of those 3 pRb sites in FDCP-Mix cells suggesting that dephosphorylated pRb participate to their quiescence. Hypoxia also induced a significant increase of the p27 KIP1 protein and mRNA in FDCP-Mix cells. Finally, we showed that the quiescent differentiated Gr-1 + cells expressed higher p27 KIP1 levels than the Gr-1 -cycling cells, a fraction containing CRA cells. That is most probably responsible of the accumulation of unphophorylated pRb. Our present data that extend personal preliminary results (39) are similar to those very recently published by Eliasson at al. [START_REF] Eliasson | Hypoxia mediates low cell-cycle activity and increases the proportion of long-term-reconstituting hematopoietic stem cells during in vitro culture[END_REF]. Altogether they show that p27 KIP1 is involved in the regulation of FDCP-Mix cell cycling in hypoxia.

The last important point of our work is that it revealed a minor stem cell-like subpopulation (that repopulates liquid cultures after returning them to 20% O 2 ) of the FDCP-mix cell line that kept its self-renewal potential after 3 to 7 days of culture at 0.1% O 2 . Similarly to normal stem cells and to cancer cells, the CRA-type FDCP-Mix cells are highly O 2 independent since they survive and proliferate in nearly anoxic conditions (0.1% O 2 ). Further experiments will explore a) the metabolic peculiarities of the low-energy proliferation mechanism of Gr-1 - FDCP-Mix cells, b) the relationship of this mechanism to self-renewal divisions uncoupled to differentiation and c) the roles of HIF-1, p27 KIP1 and pRb in the regulation of balance between quiescence and differentiation of FDCP-Mix.

Materials and methods

Maintenance of the FDCP-Mix cell line in culture

The FDCP-Mix cells (kindly provided by G. Mouchiroud ; Lyon, France) were routinely replated at 5x10 4 cells/mL every 3 days in Iscove's modified Dulbecco's medium (IMDM, Invitrogen, Carlsbad, CA, USA) complemented with penicillin (50 units/mL), streptomycin (50 µg/mL), 20% horse serum (HS, Invitrogen), and 10% Wehi conditioned medium (CM) as source of IL-3. All cultures were carried out at 37°C in a humidified 5% CO 2 incubator (Binder, Tuttlingen, Germany). For experiments, cells in exponential growth at day 3 of culture were centrifuged, washed and resuspended in complete medium containing Wehi-CM as required.

Cell proliferation, viability and apoptosis in ambient and low O 2 concentration

FDCP-Mix cells were seeded at 1x10 5 cells/mL and cultured for 24, 48 and 72h in the same culture medium either at 20% O 2 in a humidified 5% CO 2 incubator or at 0.1% O 2 concentration in an O 2 /CO 2 incubator (Xvivo, BioSpherix, Redfield, NY, USA). After culture, viable cells were counted on Malassez Cell with Trypan Blue exclusion. Apoptosis was measured using the Annexin-V-FITC Kit (#PN IM3546, Beckman Coulter, Fullerton, CA, USA) following manufacturer protocol. Samples were analyzed by flow cytometry (FCM) with Becton Dickinson FACSCanto II and FACSDiva TM software (San Jose, CA, USA).

Analysis of cell cycle and divisions by flow cytometry

Cell cycle was analyzed at 24, 48 and 72 hours of culture at 20% O 2 and at 0.1% O 2 . In order to distinguish G 0 , G 1 , S and G 2 /M cell cycle phases, cells were double labeled with Propidium Iodide (PI) (quantitative DNA labeling) and Ki-67 antibody (absent only in G 0 phase cells).

Briefly, cells washed in PBS were incubated 30 min at room temperature (RT) in a PFA/saponin solution (0.4% PFA, 10mM HEPES, 0.02% saponin, H 2 O). Cells were washed two times in "washing buffer" (5% FBS, 0.05% sodium azide, 55mg/L EDTA, 0.02% saponin) and incubated 30 min at RT with an anti-Ki-67 antibody or isotype (Alexa Fluor® 647 conjugated anti-Ki-67: #558615, Alexa Fluor® 647 conjugated isotype: #557732, BD Pharmingen, San Diego, CA, USA). Washed cells were resuspended in PBS supplemented with PI (20µg/mL) to quantitatively label DNA. Double labeled cells were then analysed by FCM using a Becton Dickinson FACSCanto II and FACSDiva TM software. In order to know if G 0 cells were issued from cells previously in G 0 and G 1 phases or from cells in S, G 2 and M phases that divided before entering in G 0 , cells were labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE; a stable vital cellular fluorescent dye whose decay allows to analyze cell divisions; #C34554, Invitrogen) according to manufacturer instructions. Briefly, 1x10 6 FDCP-Mix cells were incubated in the dark during 15min at 37°C in 1ml of prewarmed PBS + 1%BSA and 10µM CFSE. Cells were then washed in FDCP-Mix culture medium and incubated 30 min at 37°C in the dark. After a last wash, cells were resuspended in complete culture medium (3x10 5 cells/mL) and incubated (37°C, 20% O 2 , 5% CO 2 ) during 24h to eliminate dead cells and use cell populations able to proliferate. Cell aliquots were then cultured either at 20 or 0.1% O 2 for 24, 48 or 72h before being labeled with anti Ki-67 and PI as described and analyzed by FCM.

Cytospin and May-Grünwald/Giemsa staining 1.5x10 5 cells resuspended in 50µL of HS were cytospun at 300 rpm during 20min using a cytocentrifuge (Shandon CytoSpin III, GMI, Ramsey, MN, USA) dried and stained with May-Grünwald before microscopical examination at X100 and X600 magnification (AX70, Olympus, Tokyo, Japan).

Analysis of cell surface markers and cell sorting

Cells cultured at 20% O 2 and at 0.1% O 2 were washed with washing buffer, incubated 30min with fluorescein isothiocyanate or phycoerythrin conjugated Isoptype, anti Mac-1, Gr-1, CD34 and CD117 antibodies (respectively, #22225033, #22159113, #22155243, #22150344 and #22151173, Immunotools, Friesoythe, Germany). Washed cells were then analyzed by FCM using a Becton Dickinson FACSCanto II and FACSDiva TM software or sorted according to their Gr-1 expression using a Becton Dickinson FACSAria.

Analysis of p27 KIP1 expression in Gr-1 -and Gr-1 + cells

Cells were double labeled first with anti-Gr-1 (as described in the previous paragraph) and then with anti-p27 KIP1 antibody according mostly to the procedure described for cell cycle analysis. Briefly, cells were incubated for 30 min with biotinylated anti-p27 KIP1 antibody (diluted 1/300, # MS-256-B, Thermo Scientific, Rockford, IL, USA) instead of anti-Ki-67.

Cells were then washed twice with washing buffer before a final 20 min incubation with streptavidin-PE (diluted 1/1000, #IM0557, Brea, CA, USA).

Culture-Repopulating Ability (CRA) assay

After 72h of Liquid Culture (LC) at 20% or 0.1% O 2 (LC1), cells were centrifuged 5 min at 300g at RT and transferred at 1x10 5 cells/mL in fresh medium into secondary cultures always incubated at 20% O 2 , irrespective of whether LC1 had been incubated at 20% or 0.1% O 2 (non-selective LC2). Cells were then further replated every 3 days and always incubated at 20% O 2 (LC3 to LC5). At the end of each LC, cell aliquots were harvested for counting following Trypan Blue exclusion (CRA cell ) and CFA assay (CRA CFC ).

The CRA assay estimates the maintenance of the stem cell subset contained within a cell population which has undergone a treatment (for instance, incubation in hypoxia) in a selective LC1 by means of cell transfer into non-selective (for instance, normoxic) LC2 and a further incubation therein. LC2 repopulation is then measured by counting the total number of cells (CRA cell ) or the number of clonogenic (CFC) cells (CRA CFC ) by means of a CFA assay (see below). In normal hematopoietic cell populations, the standard CRA assay has been shown to detect short-term-repopulating hematopoietic stem cells [START_REF] Cipolleschi | The expansion of murine bone marrow cells preincubated in hypoxia as an in vitro indicator of their marrow-repopulating ability[END_REF].

Colony-Forming Ability (CFA) Assay

Western Blot Analysis

Cells cultured at 20 % O 2 and at 0.1% O 2 were washed in PBS and lysed in loading buffer (2% SDS, 10% glycerol, 50mM Tris pH7.8). Protein concentration was determined using the BCA Pierce assay (Thermo Scientific). Then, 40µg of whole protein cell extract were resolved in SDS-PAGE and transferred onto a PVDF Membrane (#RPN303F, GE Healthcare, Uppsala, Sweden) to probe for pRb and p27 proteins respectively. The membrane was saturated with non fat milk (5%) in TBS-T (10mM Tris pH 7.8, 150mM NaCl, Tween 0.1%) and then incubated overnight at 4°C with specific antibodies to phospho-ser 608 pRb, phospho-ser 807/811 pRb, p27 KIP1 (respectively #2181, #9308 and #2552, Cell Signaling Technology, Beverly, MA, USA), pRb (#554136, BD Pharmingen) or phospho-thr 821 pRb (#44-582, Invitrogen) diluted as indicated by the manufacturer. After 3 washes in TBS-T, membranes were incubated 1 h with antirabbit or antimouse horseradish peroxydaseconjugated antibodies. The bands were visualized using the ECL system (#WBKLS, Millipore, Billerica, MA, USA). The membrane was washed again, saturated, stained with an anti-β-actin antibody (#A5316, Sigma, St Louis, MO, USA) and finally revealed as described above.

Reverse transcription and quantitative PCR

Cells cultured at 20 % O 2 and at 0.1% O 2 were washed in PBS and treated with 250µL Trizol (#15596-026, Invitrogen). Total RNA extraction, DNAse treatment (#M6101, Promega, Madison, WI, USA) and reverse transcription with M-MLV Reverse Transcriptase (#M1705, Promega) were realized according to manufacturer's instructions. Samples were run three times in duplicates using the brilliant SYBR Green QPCR (#600548, Stratagene, La Jolla, Ca, USA) in the MX4000 apparatus (Stratagene) according to the manufacturer's instruction. The real-time PCR reactions were carried out in the presence of mouse p27 KIP1 gene specific oligonucleotide primers (forward primer: 5'AGGCGGTGCCTTTAATTGGG, reverse primer: 5'TTACGTCTGGCGTCGAAGGC) or 36B4 gene specific oligonucleotide primers (forward primer: 5'-GCTTCATTGTGGGAGCAGAC and reverse primer:

5'CATGGTGTTCTTGCCCATCAG): 10 min activation at 95°C, followed by 40 cycles at 95°C for 30 sec, 58°C for 1 min and 72°C for 1 min.

A relative quantification of p27 KIP1 transcript expression was obtained by standardizing to 36B4 levels that was shown to be stable in hypoxia [START_REF] Dayan | The oxygen sensor factor-inhibiting hypoxia-inducible factor-1 controls expression of distinct genes through the bifunctional transcriptional character of hypoxia-inducible factor-1alpha[END_REF]. The final value was calculated by taking an average of relative transcript copy numbers amplified from the 3 sets of PCR reaction performed in duplicates.

Titles and legends to figures

  parallel (fig 2b to d). To determine whether this G 0 phase accumulation was due to cells previously in G 0 and G 1 or to cells entering in G 0 after one or several divisions, FDCP-Mix cells labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE) (a stable vital cellular fluorescent dye whose decay allows to analyze cell divisions) were cultured for 72h at 20% O 2 and at 0.1% O 2 . At this time point, division rates (CFSE labeling) and cell cycle status (Ki-67 + or Ki-67 -) of cultured FSCP-Mix cells were co-analyzed by flow cytometry (FCM). As demonstrated by the comparison of panels b and c of fig 3, the division rate of FDCP-Mix cells was slower at 0.1% than at 20% O 2 . This is due to the association of two cell cycle events: a) the extension of the cell cycle duration as concluded from the respective percentages of Ki-67 + cells in each division class and b) the progressive accumulation of G 0 cells after each division (revealed by comparing the percentages of Ki-67 -cells in each division class at 20 and 0.1% O 2 ).

1x10 3 FDCP

 3 -Mix cells were seeded in 250µL of methyl-cellulose (Methocult TM , #H4230, Stemcell technologies, Vancouver, Canada) with 10ng/mL of mIL3 (#213-13, Peprotech, Rocky Hill, NJ, USA) in duplicate. Culture dishes were incubated for 11 days at 37°C in humidified atmosphere with 20% O 2 , 5% CO 2 before counting the number of colonies (aggregates >50 cells).

Figure 1 :

 1 Figure 1: Influence of O 2 concentration on the proliferation and apoptosis of FDCP-Mix cells.Cells were seeded at 1x10 5 cells/mL and cultured 24, 48 and 72h at 20, 3, 1 or 0.1% O 2 .

Figure 2 : 1 (

 21 Figure 2: Influence of O 2 concentration on the cell cycle of FDCP-Mix cells. Cells were seeded at 1x10 5 cells/mL and cultured at 20% or 0.1% O 2 for 24h, 48h and 72h. Double labeling with PI and Ki-67 antibody identifies cells (a) in G 0 (DNA =2N; Ki-67 -), (b) in G 1 (DNA =2N; Ki-67 + ), (c) in S (2N< DNA <4N; Ki-67 + ) and (d) in G 2 /M (DNA =4N; Ki-67 + ).

Figure 3 :

 3 Figure 3: Hypoxia (0.1% O 2 ) reduces the number of cell divisions of FDCP-Mix cells. Dot

Figure 4 :

 4 Figure 4: Hypoxia (0.1% O 2 ) increases granulo-monocytic differentiation of FDCP-mix cells.

Figure 5 :

 5 Figure 5: Hypoxia (0.1%) maintains a minor population of FDCP-Mix cells with Culture

Figure 6 :

 6 Figure 6: Gr-1 + and Gr-1 -FDCP-Mix cells have different morphology, CRA cell and CRA CFC

Figure 7 :

 7 Figure 7: Time dependent modifications of pRb during culture in hypoxia. After 24, 48 or 72h

Figure 8 :

 8 Figure 8: Increase of p27 KIP1 during culture of FDCP-Mix cells at 0.1% O 2 is related to their

  Figure 1

Figure 4 72h

 4 Figure 4 72h at 20% O 2 72h at 0.1% O 2 b a x100 x100 Isotype 20% O 2 20% O 2 0 1% O I t 0 1% O x100 x100

  Figure 5

2 Gr

 2 Figure 7

Aknowledgements

This work was funded by grants from la ligue nationale contre le cancer (comité sud-ouest) and association Laurette Fugain. AG, CD and FH received a doctoral fellowship from the French ministère de l'Education Nationale, de la Recherche et de la Technologie.

The present affiliation of FH is the Institut de Recherche Criminelle de la Gendarmerie Nationale, Service Central d'analyses Génétiques de la Gendarmerie, France.

The language corrections of manuscript by Mrs. Elisabeth Volkmann are highly appreciated.

Statistics

All data are presented as mean ± standard deviation of 3 to 7 independents experiments.

Friedman or Kruskal & Wallis test were respectively used to compare more than 2 experimental conditions paired or not. For statistical analysis of differences between 2 not paired experimental conditions, Mann & Whitney test were used.


M Elena Fernández-Tresguerres

Susana Moreno-Díaz De La Espina

Fátima Gasset- Rosa

Rafael Giraldo
email: rgiraldo@cib.csic.es

A DNA-promoted amyloid proteinopathy in Escherichia coli

Keywords: Escherichia coli, Protein aggregation, Protein amyloids, Proteinopathy, RepA protein Escherichia coli / Protein aggregation / Protein amyloids / Proteinopathy / RepA protein

Protein amyloids arise from the conformational conversion and assembly of a soluble protein into fibrilar aggregates with a crossed β-sheet backbone. Amyloid aggregates are able to replicate by acting as a template for the structural transformation and accretion of further protein molecules. In physicochemical terms, amyloids arguably constitute the simplest self-replicative macromolecular assemblies. Similarly to the mammalian proteins PrP and α-synuclein, the winged-helix dimerization (WH1) domain of the bacterial, plasmid-encoded protein RepA can assemble into amyloid fibers upon binding to DNA in vitro. Here we report that a hyper-amyloidogenic functional variant (A31V) of RepA, fused to a red fluorescent protein, causes an amyloid proteinopathy in Escherichia coli with the following features: i) in the presence of multiple copies of the specific DNA sequence opsp, WH1(A31V) accumulates as cytoplasmatic inclusions segregated from the nucleoid; ii) such aggregates are amyloid in nature; iii) bacteria carrying the amyloid inclusions age, exhibiting a fivefold expanded generation time; iv) before cytokinesis, small inclusions are assembled de novo and transferred to the daughter cells, in which transmission failures cure amyloidosis; and v) in the absence of inducer DNA, purified cellular WH1(A31V) inclusions seed amyloid fiber growth in vitro from the soluble protein. RepA-WH1 is a suitable bacterial model system for amyloid proteinopathies.

Introduction

Protein amyloids, aggregated conformational variants of an otherwise soluble protein, are built as cross-β structures. These are fibrilar β-sheet assemblies in which strands donated by individual protein molecules are arranged perpendicular to the axis of the fiber [START_REF] Chiti | Protein misfolding, functional amyloid, and human disease[END_REF][START_REF] Sawaya | Atomic structures of amyloid cross-β spines reveal varied steric zippers[END_REF]. Protein amyloids are the causative agents of a large number of human neurodegenerative and systemic diseases, and are therefore the subject of intense biomedical research. Protein amyloidosis can be propagated by fiber fragmentation, followed by vertical spread of the generated seeds to progeny cells, where they act as a template for the conformational conversion of soluble protein molecules and nucleate their accretion into growing fibers [START_REF] Wiltzius | Molecular mechanisms for protein-encoded inheritance[END_REF][START_REF] Knowles | An analytical solution to the kinetics of breakable filament assembly[END_REF]. Prions are a subset of amyloid proteins with the distinctive ability to be horizontally spread by cell-to-cell infection [START_REF] Krishnan | Structural insights into a yeast prion illuminate nucleation and strain diversity[END_REF][START_REF] Cobb | Prion diseases and their biochemical mechanisms[END_REF]. Bacteria, while able to secrete functional extracellular protein amyloid aggregates [START_REF] Otzen | We find them here, we find them there: functional bacterial amyloid[END_REF], do not naturally form intracellular amyloids. An exception to this rule is inclusion bodies, produced upon over-expression of aggregation-prone recombinant proteins, which exhibit some amyloid features [START_REF] Morell | Inclusion bodies: specificity in their aggregation process and amyloid-like structure[END_REF][START_REF] Wang | Bacterial inclusion bodies contain amyloid-like structure[END_REF][START_REF] Wasmer | Solid-state NMR spectroscopy reveals that E. coli inclusion bodies of HET-s(218-289) are amyloids[END_REF][START_REF] De Groot | Amyloids in bacterial inclusion bodies[END_REF]. However, an increasing number of proteins, many of bacterial origin, are being identified that can be forced to aggregate as amyloids in vitro through the induction of local unfolding under non-physiological conditions [START_REF] Chiti | Protein misfolding, functional amyloid, and human disease[END_REF]. Yeast prions such as Sup35p/[PSI+] and Ure2p/[URE3] have 3 provided a wealth of valuable information on protein amyloidosis and its transmissibility [START_REF] Wickner | Prions of fungi: Inherited structures and biological roles[END_REF][START_REF] Tessier | Unraveling infectious structures, strain variants and species barriers for the yeast prion [PSI+][END_REF]. To date no endogenous prions have been identified in bacteria.

However, bacteria are believed to posses the potential to support prion replication, since yeast [PSI+] can be heterologously propagated in Escherichia coli [START_REF] Garrity | Conversion of a yeast prion protein to an infectious form in bacteria[END_REF].

RepA protein is encoded within the replicon module of the Pseudomonas plasmid pPS10 and homologues of this protein are found in many plasmids in Gram-negative bacteria [START_REF] Giraldo | Twenty years of the pPS10 replicon: insights on the molecular mechanism for the activation of DNA replication in iteron-containing bacterial plasmids[END_REF]. RepA binds as a dimer to an operator DNA sequence to repress its own transcription. Upon binding to directly repeated DNA sequences found at the plasmid replication origin, RepA dissociates into monomers to initiate DNA replication. Similarly to the mammalian prion protein PrP [START_REF] Cordeiro | DNA converts cellular prion protein into the β-sheet conformation and inhibits prion peptide aggregation[END_REF][START_REF] Nandi | DNA-induced partial unfolding of prion protein leads to its polymerisation to amyloid[END_REF][START_REF] Deleault | RNA molecules stimulate prion protein conversion[END_REF][START_REF] Deleault | Formation of native prions from minimal components in vitro[END_REF][START_REF] Geoghegan | Selective incorporation of polyanionic molecules into hamster prions[END_REF][START_REF] Silva | Intriguing nucleic-acid-binding features of mammalian prion protein[END_REF][START_REF] Wang | Generating a prion with bacterially expressed recombinant prion protein[END_REF] and α-synuclein [START_REF] Cherny | Double-stranded DNA stimulates the fibrillation of α-synuclein in vitro and is associated with the mature fibrils: An electron microscopy study[END_REF]Hedge et al., 2010), the RepA winged-helix dimerization domain (WH1) undergoes a large α-helix to β-sheet conformational transition upon sequence-specific binding to double-stranded (ds) DNA [START_REF] Díaz-López | Structural changes in RepA, a plasmid replication initiator, upon binding to origin DNA[END_REF][START_REF] Giraldo | A conformational switch between transcriptional repression and replication initiation in the RepA dimerization domain[END_REF][START_REF] Díaz-López | Early events in the binding of the pPS10 replication protein RepA to single iteron and operator DNA sequences[END_REF]. In combination with a single point mutation in RepA (A31V), which expanded the replication host-range of pPS10 and duplicated the plasmid copy-number in vivo [START_REF] Fernández-Tresguerres | Host growth temperature and a conservative amino acid substitution in the replication protein of pPS10 influence plasmid host range[END_REF]Gasset-Rosa et al., 2008a), low-affinity and transient binding of short dsDNA sequences in vitro enhanced the conformational conversion of WH1, promoting protein amyloidosis [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF]. Thus, DNA is not a major structural component of the resulting amyloid fibers but acts as a real inducer of WH1(A31V) amyloidosis under quasiphysiological conditions [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF]Gasset-Rosa et al., 2008b).

To challenge our understanding of the molecular basis of RepA-WH1 amyloidosis in vitro, we successfully engineered amyloid assemblies in E. coli using this protein as a chassis. We found that a fusion of RepA-WH1(A31V) to a monomeric red fluorescent protein forms intracellular aggregated amyloid inclusions that are transmitted to the bacterial progeny by seeding and self-templating, causing a marked decrease in cell fitness. The RepA-WH1 aggregated inclusions appear to be the causative agent of a synthetic amyloid proteinopathy in bacteria.

Results

DNA-promoted amyloidosis of RepA-WH1 in E. coli

The DNA sequences coding for either the wild-type (WT) RepA-WH1 or its enhanced amyloidogenic, functional mutant variant A31V [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF], were cloned as 5' fusions to mCherry (mRFP), a monomeric DsRed fluorescent protein [START_REF] Shanner | Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein[END_REF], in a multicopy, IPTGinducible expression vector. A parallel vector series was constructed carrying 18 tandem repeats of the 11 bp DNA sequence opsp, which specifically promotes WH1 amyloidosis in vitro [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF]Gasset-Rosa et al., 2008b), cloned immediately downstream of the WH1-mRFP fusions. The aim of this experimental design was to facilitate the coupling between protein biosynthesis and binding to [START_REF] Morris | Toward a biomechanical understanding of whole bacterial cells[END_REF][START_REF] Montero-Llopis | Spatial organization of the flow of genetic information in bacteria[END_REF] (Fig. 1A). When these plasmids were transformed into E. coli MC4100 cells (a commonly used K-12 strain of this bacterium), two-fold higher transformation efficiencies were observed for the WT than for the A31V constructs. Moreover, all of the WH1(WT) colonies exhibited an intense red-purple color, whereas 10% of those carrying the WH1(A31V) variant were white and larger in size, while the remaining 90% were of a similar size to the WT colonies but were pale red in color (Fig. 1B). Similar results were obtained with other E. coli K-12 strains. When the plasmid contents of several colonies of each type were analyzed (Fig. 1C), it became evident that the purified expression vectors from the red colonies were essentially the same, apart from the WT or A31V inserts. However, plasmids isolated from the white colonies exhibited increased copy-numbers (by 2.5-3 fold) and sizes. Restriction analysis showed that white colonies were carrying plasmids over 1 kbp larger than expected. DNA sequencing revealed, in every plasmid analyzed, the insertion of an IS10 mobile genetic element [START_REF] De Palmenaer | IS4 family goes genomic[END_REF] interrupting the WH1(A31V)-mRFP coding sequence (Fig. S1). Insertion of this genetic element knocked out the expression of the fusion protein, as confirmed by Western blotting (Fig. 1D). These findings reflected the remarkable genome plasticity in response to stress and were the first indication of the differential toxicity linked to the amyloidogenic A31V variant of WH1-mRFP (see next section). Therefore, in all subsequent experiments single red colonies were used to inoculate culture medium.

Fluorescent microscopy analysis of the bacteria transformed with WH1(WT)-mRFP revealed that IPTG addition resulted in the emission of red fluorescence, with variable intensities, diffused across the whole cytoplasm, in about 25% of the cells (Fig. 2A). The bi-modal, all-or-nothing, expression levels achieved by the individual cells could be due to the stochastic variability intrinsic to transcription and translation [START_REF] Davidson | Individuality in bacteria[END_REF][START_REF] Robert | Pre-dispositions and epigenetic inheritance in the Escherichia coli lactose operon bistable switch[END_REF]. Expression of the WH1(A31V)-mRFP variant led to the appearance of a couple of intense fluorescent foci, commonly located around one-quarter and three-quarters of the way along individual cells, in 17% of the bacteria. These foci resembled inclusion body protein aggregates [START_REF] García-Fruitós | Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins[END_REF]. DAPI staining showed that these WH1(A31V)-mRFP inclusions were segregated from the bacterial nucleoid.

However, when extra opsp repeats were cloned in cis, allowing for translation-coupled binding of WH1 to DNA with the multiplier effect of the high copy number (40-250 per cell) of the vector, a further 40% increase in the number of cells with fluorescence labeling was achieved (Fig. 2A,right).

In addition, the foci observed in the presence of additional copies of the opsp repeats were larger than those observed in their absence (0.93 ± 0.15 µm 2 vs. 0.52 ± 0.08 µm 2 , respectively), but the fluorescence intensities, when corrected for the size of the inclusions, were similar (0.89 ± 0.26 vs. 0.85 ± 0.20, respectively). In all of the subsequent in vivo experiments, the opsp DNA repeats were included to enhance the accretion of WH1(A31V) molecules into the inclusions.

BTA-1 is a fluorophore related to thioflavin T (Th-T) that is specific for amyloid cross-β structures, but possesses a superior ability to permeate cell membranes with no interference from DNA cross- [START_REF] Cordeiro | DNA converts cellular prion protein into the β-sheet conformation and inhibits prion peptide aggregation[END_REF]and our own observations). These properties are derived from its neutral rather than positive charge [START_REF] Wu | The binding of thioflavin T and its neutral analog BTA-1 to protofibrils of the Alzheimer's disease Aβ(16-22) peptide probed by molecular dynamics simulations[END_REF]. In bacteria expressing WH1(A31V)-mRFP, BTA-1 fluorescence co-localized with the core of the protein inclusions (Fig. 2B) showing their intrinsic amyloid nature.

RepA-WH1 amyloids impair cell fitness and are transmissible

To test whether the fluorescent intracellular inclusions were detrimental to bacterial fitness, timelapse microscopy was performed. Consistently, cells exhibiting diffuse fluorescence (WT protein over-expressed) showed 1.7 fold increased generation times compared with non-fluorescent bacteria (Movie S1A), whereas cells carrying the fluorescent foci (A31V variant over-expressed) showed fivefold increased generation times (Movie S1B). The inclusion-free cells therefore became the major component of the resulting micro-colonies, compatible with the observation of weaker red coloration of A31V colonies compared with WT colonies (Fig. 1B).

The detailed observation of how bacteria containing WH1(A31V)-mRFP inclusions divide (Figs. 3 andS2) revealed that 20-30 min before any equatorial constriction separating the two daughter cells (a morphological indication for cytokinesis) became evident, a small extra cytoplasmatic fluorescent particle appeared (arrows). In cells starting with two large sub-polar inclusions (the most common situation), the emergent fluorescent particle tended to locate towards the mid-cell, close to where the new cell pole will be located (Fig. 3, cells labeled A and B''). At the point of division, the central amyloid seed had grown in to a full size protein inclusion, temporally hampering septation and resulting in elongated cells (Fig. 3, cells A' and A''). Cells overcame division arrest through the asymmetric inheritance of the central inclusion (see below) so that filaments composed of undivided bacteria were not observed, except after long incubation times. Another type of cell bore both subpolar and mid-cell inclusions (Fig. 3, cell B). In this case, the emergent fluorescent particle located at the empty pole and a subsequent asymmetric cytokinesis resulted in daughter cells carrying either a single (Fig. 3, cell B') or two sub-polar (cell B'') inclusions.

The fate of cells with a single aggregate deserves further attention (Fig. S2). If the inclusion was centered (cell A), cytokinesis asymmetrically distributed the maternal inclusion to one of the daughter cells, where it had a sub-polar location (cell A'). The transmission of amyloidosis to the other daughter cell was thus compromised, depending on the spontaneous, DNA-promoted, appearance of a seed (cell A''). This became a source of cured cells devoid of amyloid inclusions. If the single protein inclusion had a sub-polar location (Fig. S2, cells B and A') the emergent particle appeared at the opposite subpole, which continued spreading amyloidosis (cell B'). On the other hand, the daughter cell (B'') carrying an inclusion close to the pole, which had already undergone two divisions, remained quiescent. Expanded generation times and quiescence of old pole-bearing cells are both aging-related phenotypes in E. coli cells carrying inclusion bodies [START_REF] Stewart | Aging and death in an organism that reproduces by morphologically symmetric division[END_REF][START_REF] Lindner | Asymmetric segregation of protein aggregates is associated with cellular aging and rejuvenation[END_REF];

Ultrastructural features and distribution of RepA-WH1 intracellular amyloid inclusions

To study the fine structure of the intracellular amyloid inclusions, they were analyzed by confocal laser microscopy (Fig. 4A) and immuno-electron microscopy (iEM) on thin cell sections (Fig. 4B).

The fusion proteins were first targeted with an antibody against their N-terminal hexahistidine tags (Fig. 1A), and then located using secondary antibodies conjugated either to a green fluorochrome or to colloidal gold nano-particles, respectively. Confocal sections through the cells bearing WH1(WT)-mRFP revealed fluorescent spots throughout the cytoplasm that, in the case of the A31V variant, clustered into the inclusions. In iEM, the WH1(WT)-mRFP cells also exhibited the label dispersed throughout the cytoplasm (Fig. 4B, top). However, the electron-dense intracellular inclusions were selectively labeled when the WH1(A31V)-mRFP amyloidogenic variant was expressed (Fig. 4B, bottom). Cells of this type mostly exhibited the small amyloid seeds (Figs. 3,S2 and 4A) as electrondense, gold-labeled particles embedded in the less dense nucleoid domain (Fig. 4B, bottom; arrow in panel b). Mature amyloid inclusions were segregated from the nucleoid (identified by its nucleoprotein fibers; see also DAPI staining in Fig. 2A).

Purified intracellular inclusions template RepA-WH1 amyloidosis in vitro

The amyloidogenic potential of the intracellular protein inclusions was then explored in vitro. About 50% of the E. coli expressed WH1(A31V)-mRFP protein were detected in the soluble lysate fraction, the other 50% had formed aggregates (Fig. S3A). The aggregates were purified via a protocol involving washing with detergents, extensive digestion with nucleases and sucrose gradient sedimentation. The final preparation was analyzed by means of transmission electron microscopy (TEM; Fig. S3B) and its amyloid nature was confirmed by Th-T binding (Fig. S3C).

Sub-stoichiometric amounts of the purified WH1(A31V)-mRFP aggregates were added to either the WT or the A31V variant of soluble RepA-WH1 under in vitro amyloidogenic conditions optimal for the slow and ordered growth of fibers [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF], but lacking the inducer, opsp-dsDNA.

Aliquots were taken at time intervals throughout the incubation and the progression of protein amyloidosis was tracked by Th-T binding and analysis of fluorescence emission (Fig. 5A). The WH1(A31V)-mRFP aggregates, when compared with the samples incubated in their absence, accelerated the appearance of amyloid material by one week. It is noteworthy that seeding was exerted on both the homologous (A31V) and the heterologous (WT) WH1 variants, although for the latter to a more reduced extent, compatible with its milder amyloid character (Giraldo, 2007). TEM inspection of the final aggregates (Fig. 5B) revealed that, whereas both unseeded WH1 proteins produced small granular particles, the addition of WH1(A31V)-mRFP seeds resulted in hallmark large [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF]. The latter neatly emerged from the WH1(A31V)-mRFP inclusions and were indistinguishable from the standard amyloid fibers promoted by opsp-dsDNA [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF]Gasset-Rosa et al., 2008b) (Fig. 5B), indicating that once DNA has promoted the conformational transformation of WH1(A31V) in vivo this is preserved to template amyloid growth in vitro. These results are consistent with the evidence presented above on mother cell-to-daughter cell seed transmission, rather than stochastic growth of a nucleus from the soluble protein pool, as the mechanism for efficient spreading of RepA-WH1 amyloidosis in vivo.

Discussion

In this study we show that the expression of a hyper-amyloidogenic mutant variant (A31V) of a plasmid DNA replication protein (RepA-WH1) fused to a red fluorescent reporter (mCherry/mRFP) results in the generation of amyloid protein aggregates exhibiting a toxic phenotype in E. coli, which is evident by a large expansion in the generation time of bacteria. Such amyloid inclusions are vertically transmitted to the progeny (i.e., coupled to cell division). Purified WH1(A31V)-mRFP aggregates can template in vitro the growth of amyloid fibers from soluble WH1(A31V) protein. These fibers are morphologically identical to the fibers grown using dsDNA as a cofactor [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF]Gasset-Rosa et al., 2008b), indicating the close relatedness of these two types of amyloid assemblies.

A significant feature of the RepA-WH1(A31V) protein is the striking ability of small dsDNA ligands of defined sequence (opsp) to promote, through transient binding, the assembly of the protein into amyloid fibers in vitro [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF]Gasset-Rosa et al., 2008b). In terms of the ensemble concept for native protein structures [START_REF] Boehr | The role of dynamic conformational ensembles in biomolecular recognition[END_REF], allosteric binding of dsDNA to WH1 [START_REF] Díaz-López | Structural changes in RepA, a plasmid replication initiator, upon binding to origin DNA[END_REF] would be determinant in selecting a subset of aggregation-prone protein conformations among those fluctuating at equilibrium, thus driving the whole process towards amyloid assembly. We have successfully conferred the ability of DNA to promote WH1(A31V) amyloidosis in E. coli cells, by showing that cloned repeats of the specific opsp dsDNA sequence enhance protein accretion to the intracellular inclusions in vivo (Fig. 2A, right). It is remarkable that, allowing for a single mismatch, the E. coli genome (http://genolist.pasteur.fr/Colibri) carries up to 48 copies of the opsp sequence, which seem able to trigger WH1(A31V) aggregation in the absence of plasmid-borne opsp repeats, although with a reduced frequency (Fig. 2A,left). It follows that substoichiometric (catalytic) levels of inducer DNA would be sufficient to promote amyloidosis in the presence of a vast excess of WH1(A31V) molecules. The presence of multiple dispersed copies of the opsp sequence in the E. coli genome made it unfeasible to study WH1 amyloidosis in the absence of a specific inducer ligand. The potential of nucleic acids to act as promoters of amyloidogenesis in vitro has been extensively documented for proteins involved in neurodegenerative amyloid diseases, such as PrP prion [START_REF] Cordeiro | DNA converts cellular prion protein into the β-sheet conformation and inhibits prion peptide aggregation[END_REF][START_REF] Nandi | DNA-induced partial unfolding of prion protein leads to its polymerisation to amyloid[END_REF][START_REF] Deleault | RNA molecules stimulate prion protein conversion[END_REF][START_REF] Geoghegan | Selective incorporation of polyanionic molecules into hamster prions[END_REF] [START_REF] Deleault | Formation of native prions from minimal components in vitro[END_REF][START_REF] Deleault | Species-dependent differences in cofactor utilization for formation of the protease-resistant prion protein in vitro[END_REF][START_REF] Wang | Generating a prion with bacterially expressed recombinant prion protein[END_REF][START_REF] Abid | Cellular factors implicated in prion replication[END_REF]. These findings underline the importance of studying amyloidogenesis in a biologically relevant context [START_REF] Bellotti | Amyloidogenesis in its biological environment: Challenging a fundamental issue in protein misfolding diseases[END_REF]. This is the first time that the inducer effect of nucleic acids, via RepA-WH1(A31V) amyloidosis, has been shown both in vitro and in vivo. It is noteworthy that the same protein sequence stretch directly contributing to RepA-WH1 amyloidosis [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF] was recently found to be involved in establishing the RepA-RepA assemblies that negatively regulate plasmid replication (Gasset-Rosa et al., 2008a). However, direct evidence for an amyloid structure in such assemblies is lacking. Although most amyloidoses described to date involve cytoplasmatic protein deposits, the first nuclear-localized yeast prion has just been reported for the transcription factor Sfp1p/[ISP+] [START_REF] Rogoza | Non-Mendelian determinant [ISP+] in yeast is a nuclear-residing prion form of the global transcriptional regulator Sfp1[END_REF]. The general biomedical relevance of our bacterial model system for DNA-promoted protein amyloidosis may be more farreaching, since protein amyloid deposits in the cell nucleus are commonly associated with human neurodegenerative diseases caused by proteins bearing polyglutamine repeats, as is the case for Huntington's disease [START_REF] De Rooij | Subcellular localization of the Huntington's disease gene product in cell lines by immunofluorescence and biochemical subcellular fractionation[END_REF] and diverse spinocerebellar ataxias [START_REF] Bauer | The pathogenic mechanism of polyglutamine diseases and current therapeutic strategies[END_REF], and is also true for transmissible spongiform encephalopathies [START_REF] Mangé | Scrapie-like prion protein is translocated to the nuclei of infected cells independently of proteasome inhibition and interacts with chromatin[END_REF]. A role for nucleic acids in promoting protein amyloidosis in these diseases cannot therefore be excluded.

Regarding the intracellular distribution of WH1(A31V)-mRFP amyloid particles, similar fluorescent spots have previously been described for multicopy plasmids lacking a partition system, when labeled with fluorescent protein reporters tightly bound to DNA [START_REF] Pogliano | Multicopy plasmids are clustered and localized in Escherichia coli[END_REF][START_REF] Anand | Plasmid segregation: Birds of a feather try not to flock together[END_REF]. However, such plasmid-linked foci usually have better defined circular contours and are smaller in size than the WH(A31V) inclusions. It is unlikely that the expression plasmids used in our study would determine the clustering of protein aggregates, thus acting as a physical carrier for the transmission of amyloid particles because, whereas WH1(WT)-mRFP appeared dispersed throughout the cytoplasm, WH1(A31V)-mRFP aggregated into inclusions (Fig. 2A), despite identical DNA binding properties between the two proteins (low affinities and high off-rates) [START_REF] Díaz-López | Early events in the binding of the pPS10 replication protein RepA to single iteron and operator DNA sequences[END_REF]Gasset-Rosa et al., 2008a;[START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF]. It is likely that the large fraction of volume occupied by the nucleoid is the main determinant, through physical exclusion, of the distribution of protein aggregates throughout the cytoplasm [START_REF] Winkler | Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing[END_REF]. Whether other factors, such as DnaK-J and ClpB chaperones [START_REF] Rokney | E. coli transports aggregated proteins to the poles by a specific and energy-dependent process[END_REF][START_REF] Winkler | Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing[END_REF], are involved in DNA-promoted RepA-WH1 amyloidosis by generating transmissible seeds through the fragmentation of intracellular inclusions, as is the case for their eukaryotic orthologues Hsp70-40s and Hsp104 in the spreading of prions in yeast [START_REF] Shorter | Hsp104, Hsp70 and Hsp40 interplay regulates formation, growth and elimination of Sup35 prions[END_REF][START_REF] Tyedmers | Prion induction involves an ancient system for the sequestration of aggregated proteins and heritable changes in prion fragmentation[END_REF], remains to be determined. It is possible that due to the small size of the transmitted WH1(A31V)-mRFP particles they could evade detection by conventional fluorescence microscopy, as it is the case for yeast propagons which are generated in large numbers [START_REF] Byrne | The number and transmission of [PSI+] seeds (propagons) in the yeast Saccharomyces cerevisiae[END_REF][START_REF] Sindi | Prion dynamics and the quest for the genetic determinant in protein-only inheritance[END_REF]. However, we observe a discrete number (usually one) of emergent small WH1(A31V)-mRFP particles in the newly formed poles of bacterial cells (Figs. 3 andS2), which then grow to the size of mature inclusions. To our knowledge, the toxicity of the WH1(A31V)-mRFP inclusions, in terms of their effect on the rate of cell division, is the highest reported for any inclusion body (IB) in E. coli to date [START_REF] Stewart | Aging and death in an organism that reproduces by morphologically symmetric division[END_REF][START_REF] Lindner | Asymmetric segregation of protein aggregates is associated with cellular aging and rejuvenation[END_REF][START_REF] Winkler | Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing[END_REF]. IBs are bacterial intracellular protein aggregates commonly considered to be inert deposits. However, the presence of protein molecules within these IBs retaining a functional conformation is feasible [START_REF] Martínez-Alonso | Learning about protein solubility from bacterial inclusion bodies[END_REF]. The pathways through which the WH1(A31V)-mRFP inclusions exert their toxicity will be the subject of future research and might provide new insight into molecular mechanisms common to other proteinopathies.

The amyloid character of the intracellular inclusions described in this study, their cell-to-cell transmissibility and their ability to nucleate and act as templates for the assembly of fibers, qualify RepA-WH1 as a minimalist model system to study the determinants of protein amyloidogenesis in general and, due to its associated toxicity, more specifically of amyloid proteinopathies. Furthermore, the fully synthetic nature of RepA-WH1 amyloids makes them suitable candidates for the bottom-up design of more complex biological assemblies [START_REF] Deplazes | Piecing together a puzzle: An exposition of synthetic biology[END_REF][START_REF] De Lorenzo | Synthetic biology: Something old, something new[END_REF]. Although the RepA-WH1 amyloids described here are transmissible, they clearly differ from yeast prions in lacking both infectivity and a selectable, gain-of-function epigenetic phenotype [START_REF] Wickner | Prions of fungi: Inherited structures and biological roles[END_REF][START_REF] Tessier | Unraveling infectious structures, strain variants and species barriers for the yeast prion [PSI+][END_REF]. Engineering these desirable characteristics into RepA-WH1 amyloids is a future target for synthetic biology in this field.

Experimental procedures WH1-mRFP +/-(opsp) 18 gene fusions

Two oligonucleotides containing the opsp-dsDNA sequence (bold) [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF], 5'-CAATGACAAGTCAATGACAAGT (opsp-A) and TGTTCAGTTACTGTTCAGTTAC-5' (opsp-B), were phosphorylated (T4-PNK), annealed and ligated (T4-DNA ligase) to build concatemers.

After end-filling (Klenow DNA polymerase), the ligation product ladders were visualized on 2% agarose gels. Concatemers were cloned into the expression vector pRGrectac-NHis-WH1 (WT and A31V, for short pWH1(WT/A31V); [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF], previously linearized with SmaI. The monomeric variant of DsRed mCherry was amplified by PCR (Pfu DNA polymerase) from the plasmid pRSETb-mCherry [START_REF] Shanner | Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein[END_REF] using the following primers (mutations in upper case):

BspEI 5' agctcaagGgaagctCTGGTTCATCCGGAatggtgagcaagggcgagga Q L K G S S G S S G M V S K G E E -WH1 -|------linker ------|-----mCherry ----- 5' agtcgacctgcaggcatgcaAGCTtCacttgtacagctcgtccatgc HindIIIEND K Y L E D M G --------pUC18 mcs --------|-----mCherry -----
The PCR product was fused downstream of repA-WH1 in pWH1(WT/A31V), either with or without the cloned (opsp) 18 repeats (see above). Plasmids were linearized with HindIII and then recombined with the PCR fragment using the In-Fusion Dry-Down PCR cloning kit (Clontech). The

Expression of WH1-mRFPs

All in vivo experiments were carried out in the common E. coli K-12 strain MC4100 [START_REF] Ferenci | Genomic sequencing reveals regulatory mutations and recombinational events in the widely used MC4100 lineage of Escherichia coli K-12[END_REF]. pLacI, a pACYC184 (Cm R ) derivative containing a cloned 1212 bp BstYI (lacI) fragment, was used as a donor for the LacI q repressor. Bacteria grown overnight on LB-agar plates plus ampicillin (100 µg.mL -1 ) were then inoculated in the same (liquid) medium and grown at 37ºC to early exponential phase (OD 600 = 0.2). The cells were then induced with 0.5 mM IPTG for 2 h. Expression analysis was carried out by SDS-PAGE, followed by Western-blot detection of the His6-tagged WH1-mRFPs. This was performed using an anti-His murine monoclonal primary antibody (1:5,000 dilution; Sigma) and a secondary anti-mouse HRP-conjugated antibody (1:10,000) and chemiluminescent detection (ECL plus; GE Healthcare). After expression, plasmids from 5x10 9 cells (1.0 OD 600 ≈ 1.5×10 9 cells.mL -1 ) were purified (High Pure Plasmid Isolation kit; Roche) and then analyzed by UVabsorption spectrophotometry and agarose gel electrophoresis.

Fluorescence microscopy

Routine microscopic observation of the bacterial cells was performed using a live cell imaging AF6000 LX equipment (Leica), consisting of a DM16000B inverted microscope, a HCX PLAN APO (100× / NA 1.40) oil immersion objective and a Hamamatsu C9100-02 CCD camera. The following excitation and suppression filters were used: mCherry (BP 546/12, BP 600/40); DAPI (BP 360/40, BP 470/40) Alexa 488 (BP 480/40, BP 527/30); and BTA (BP 467/37). Phase contrast images of the fields were also recorded. Confocal laser microscopy was carried out using a Leica imaging device (TCS-SP2-AOBS, including a DMIRE2 inverted microscope). The software provided by Leica was used for the superposition of the views resulting from the illumination of a given field at different regions of the spectra and for image quantifications.

For observations performed on fixed bacteria, 0.5-1.0 mL culture aliquots were spun-down and the cells were washed with the same volume of PBS buffer (10 mM Na 3 PO 4 , 150 mM NaCl, 15 mM KCl pH 7.4). Cells were collected by centrifugation at 1,600 g and the cell pellet was then fixed in fresh 4% paraformaldehyde (PFA) in PBS buffer, for 30 min at room temperature. After fixation, the cell pellets were washed twice in PBS. In some experiments, cells were stained with 5 µg.mL -1 DAPI (4´,6-diamidino-2-phenylindole.2HCl; Serva) for 15 min during fixation. 10 µL aliquots of cells in PBS were air-dried on poly-L-lysine coated slides and mounted with Vectashield (Vector Laboratories). For BTA-1 staining, cells were fixed in PFA and washed in PBS, as described above. Then they were suspended in BTA (2-(4´methylaminophenyl) benzothiazole (Sigma); 1 mM in ethanol), incubated for 30 min at room temperature, washed in PBS and mounted.

For immunofluorescent staining, cells were fixed in freshly prepared 2% PFA, made in PBS buffer containing 0.5% Triton X-100, for 30 min at room temperature. After three washes with the same solution, pellets were suspended in GTE buffer (50 mM glucose, 10 mM EDTA, 20 mM Tris . HCl pH 7.5). Then, 15 µL aliquots were air-dried on poly-L-lysine coated slides, incubated with freshly prepared lysozyme (8 µg.mL -1 in GTE), for 5 min at room temperature, and washed twice with PBS for 10 min. Next, they were incubated with blocking solution (PBS plus 0.05% Tween-20, 2% BSA), for 30 min at room temperature. Slides were then incubated with the anti-His monoclonal primary antibody (1:400 dilution in blocking solution), at 4ºC overnight. The preparations were then washed five times for 10 min in PBS containing 0.05% Tween-20 and incubated with Alexa 488-coupled antirabbit secondary antibody (Molecular Probes; 1:100 dilution in blocking solution) for 1 h at room temperature in the dark. After five washes with PBS plus Tween-20, samples were air-dried and mounted as described above. Negative controls were performed omitting the incubation with the primary antibody.

For time-lapse microscopy of living bacteria, 100 µL of melted aerated 1.5% agar-LB (supplemented with ampicillin) were placed onto a glass slide, overlaid with a coverslip and left to solidify. Then the coverslip was removed, IPTG was added and 10 µL of the cultures were immediately placed onto the agar layer and covered with a clean coverslip. Specimens were maintained in the microscope incubation chamber, under controlled humidity and temperature (37ºC), throughout all the experiment. Images were digitally captured at the specified time intervals.

Transmission Electron Microscopy (TEM) and immuno-Electron Microscopy (iEM)

Purified protein inclusions and aggregates assembled in vitro were examined after negative staining with uranyl acetate, as indicated previously [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF]. TEM was carried out using a JEOL JEM-1230 transmission electron microscope, operated at 100 kV. Photographic films (Kodak) were digitized using an AGFA Arcus 1200 scanner.

For iEM, cultures of exponentially growing cells were fixed in 2% PFA in PBS buffer for 1h, washed three times in PBS for 10 min and then embedded in 2% agarose. The agarose blocks were cut into small pieces, washed in PBS, dehydrated through an ethanol series (from 30% to absolute ethanol), embedded in London white resin and cured at 60ºC. Immuno-gold labeling was performed on ultra-thin sections of cells collected on the top of Formvar-coated nickel grids. These were successively floated on drops of: 20 mM glycine in PBS for 15 min; PBS containing 0.05% Tween-20 for 10 min; blocking solution (PBS plus 0.05% Tween-20, 2% BSA) for 30 min; and anti-His primary antibody (1:400 dilution in blocking solution), for 2 h at room temperature. After three washes (10 min each) in PBS-Tween-20, grids were incubated with an anti-mouse secondary antibody conjugated with Tween-20 and once in bi-distilled water. Grids were then air-dried and contrasted with 5% aqueous uranyl acetate for 30 min, washed in bi-distilled water and air-dried before being observed by EM (see above). Negative controls were performed omitting the incubation with the primary antibody.

Purification of soluble and aggregated WH1(A31V)-mRFP

Two Erlenmeyer flasks (2 L vol) with 400 mL of LB medium plus ampicillin (100 µg.mL -1 ) were inoculated with E. coli MC4100 cells carrying pWH1(A31V)-mRFP+opsp 18 . Cultures were grown at 30ºC to OD 600 = 0.8, when IPTG was added to 0.5 mM and the temperature was shifted to 37ºC. The cells were then incubated overnight (OD 600 = 4.0). Bacteria were harvested, washed with cold 0.9% NaCl and the cell pellet was resuspended in 15 mL of a buffer containing 1.0 M NaCl, 20 mM Tris . HCl pH 7.5, 1 mM EDTA, 1% Brij-58 (Sigma), 1 mM pNH 2 -benzamidine and 10% glycerol.

Lysozyme (1 mg) was then added and the suspension was incubated at 37ºC for 30 min before sonication (two cycles, 30 s each, using a 1 cm wide tip). The resulting cellular lysate was clarified by ultracentrifugation at 20,000 rpm (1 h, 4ºC) in a Beckman Ti60 rotor. Approximately 50% of WH1(A31V)-mRFP was detected as soluble protein in the supernatant and was purified by immobilized metal affinity chromatography (Ni 2+ -IMAC), as indicated for WH1(A31V) [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF]. Aggregated WH1(A31V)-mRFP protein was resuspended in 15 mL of a buffer including 0.1 M NaCl, 20 mM Tris . HCl pH 7.5, 10 mM MgCl 2 , 0.5% Na-deoxycholate, 0.5% N-dodecyl-N,Ndimethylammonio-3-propanesulfonate (SB-12), 1 mM DTT, 1 mM pNH 2 -benzamidine and 10% glycerol. The suspension was sonicated and centrifuged as before. The pellet was resuspended in 15 mL of the same buffer (excluding the detergents), supplemented with 5 units of DNaseI (Worthington) and 0.5 mg of RNaseA (Roche), and then incubated for 1 h at 37ºC. The protein suspension was sonicated and centrifuged again and the pellet was resuspended in 10 mL of 0.1 M Na 2 SO 4 , 4 mM MgSO 4 , 20 mM Tris . HCl pH 8 and 10% glycerol. After a further sonication/centrifugation round, aggregated protein was homogenized in 1.5 mL of the same buffer. 100 µL aliquots of this suspension were layered on sucrose discontinuous gradients (60-50-40-30-20% in the same buffer, 200 µL of each solution) cast in 2 mL Eppendorf tubes as described previously [START_REF] Liebman | Biochemical and genetic methods for characterization of [PIN+] prions in yeast[END_REF]. The tubes were centrifuged at 7,500 rpm for 16 h at 4ºC, then 100 µL aliquots were carefully removed from the top of the tube and 10 µL samples were analyzed by SDS-PAGE. WH1(A31V)-mRFP inclusions were found to peak at fraction 8 in the gradient. The protein concentration in the inclusions was estimated by dissolving 10 µL of the purified aggregates in 6 M Gu . HCl and 20 mM Tris . HCl pH 8, determining the absorbance in the UV-Vis spectrum and then using the equation C (mg.mL -1 )= 1.55 × A 280 -0.76 × A 260 [START_REF] Simonian | Spectrophotometric and colorimetric determination of protein concentration[END_REF]. From the A 280 /A 260 ratio (1.82), purified protein inclusions were judged to be free of nucleic acids.

Fluorescence spectroscopy analysis of the binding of thioflavin-T (Th-T) to amyloids

To confirm for the amyloid nature of the purified protein inclusions, 50 µL of a 4 mg.mL -1 suspension were added to 440 µL of a buffer including 0.1 M Na 2 SO 4 , 4 mM MgSO 4 and 20 mM Tris . HCl pH 8. Then 10 µL of a 3 mM Th-T (Sigma) stock (final concentration: 60 µM) were added.

To study the kinetics of amyloid seeding by the purified inclusions, 450 µL of buffer and 10 µL of the Th-T stock were supplemented with 40 µL of the amyloidogenic samples at the indicated times (0, 1 h and 1, 7, 14, 21, 28 and 35 days). This was performed as described previously [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF]; briefly, 100 µL aliquots including 35 µM of purified soluble WH1(WT) or WT(A31V) in 0.1M Na 2 SO 4 , 30 mM Tris . HCl pH 8, 4 mM MgSO 4 , 7% PEG4000 and 3% MPD were supplemented (or not, as indicated) with 1 µg of pure WH1(A31V)-mRFP inclusions. It is noteworthy that inducer opsp-dsDNA, an absolute requirement for ordered fibers [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF], was absent from the incubations.

In all cases, samples incubated with Th-T were placed in Hellma F-QS cuvettes (2×10 mm path length) and allowed to equilibrate at room temperature for 5 min before analysis. A Jobin-Ivon SPEX Fluorolog-3 spectrofluorometer, with the temperature controlled at 22ºC by a Peltier module, was used for spectra acquisition. The excitation wavelength was set to 440 nm and the excitation and emission slits were set to 5 and 10 nm, respectively. The integration time was 1 s and the spectra were acquired in 1 nm steps. Data was plotted and analyzed using KaleidaGraph v.3.6 software (Synergy Software, CA, USA). C. Plasmids purified from equivalent cell numbers in three independent IPTG-induced cultures (labeled 1-3), were quantified by UV light absorption (left), analyzed by agarose gel electrophoresis of their supercoiled forms (1/15 of each DNA preparation; right) and digested with HindIII (bottom), a single cutter restriction enzyme that renders a defined banding pattern for each plasmid. Asterisks mark the restriction-resolved plasmid multimers from the cultures inoculated with white colonies (see B). Also, note their sizes, > 1 kbp larger than the parental pWH1(A31V)-mRFP, and the pLacI band visible just below. D. SDS-PAGE analysis of total lysates (5×10 8 cells / lane) from un-induced (t = 0 h) and IPTGinduced (t = 2 h) cultures. Western-blots performed with a monoclonal antibody against the His6-tag mRFP aggregates (Fig. S3). Samples were incubated as described previously [START_REF] Giraldo | Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures[END_REF], without inducer dsDNA. Aliquots were taken at the indicated intervals, incubated with Th-T (60 µM) and the fluorescence emission spectra were acquired (λ excit = 440 nm). Emission at 481 nm was plotted against time (error bars: SD; three replicates).

Figure legends

  sequences in the diffusion-limited, crowded, bacterial cytoplasm

  intracellular distributions, and elicit less acute cellular toxicities, than the WH1(A31V)-mRFP amyloid inclusions reported here.

  WT) and well defined fibers (A31V)

  frame fusions included a (Gly-Ser-Ser) 2 -Gly flexible linker between the two protein partners. Constructs were CaCl 2 -transformed in E. coli JM109 (recA) cells and checked by restriction analysis and automated DNA sequencing (http://www.secugen.es/).

  particles (Sigma; 1:50 dilution in blocking solution), and then washed three times in PBS-

Fig. 1 .

 1 Fig. 1. Expression of WH1-mRFPs in E. coli MC4100 cells. A. Linear sketch of the plasmids used for WH1-mRFP over-expression under the tac promoter. WT or A31V (*) WH1 variants, and the cis (opsp) 18 repeats are indicated. B. Images of LB-ampicillin agar plates showing bacterial colonies transformed with plasmids pWH1(WT)-mRFP (left) and pWH1(A31V)-mRFP (right).

  mRFPs are aligned below. Different exposure times were required for detection of the basal (pre-induction) and post-induction expression levels.

Fig. 2 .

 2 Fig. 2. RepA-WH1(A31V) fused to mRFP accumulates as amyloid inclusions in the E. coli cytoplasm. A. Images of the E. coli MC4100 cells transformed with the plasmids outlined in Fig. 1A, after a 2 h induction with IPTG. Cells were fixed and the DNA was stained with DAPI. Representative microscopy fields are shown, overlaying images obtained after excitation at 546 nm (red emission, mRFP) and 360 nm (blue emission, DAPI). At the lower left corner of each panel, the decimal proportion of red fluorescent cells / total number of cells counted is shown. B. (a) Phase contrast image of induced A31V-mRFP cells, after staining with 1 mM of the amyloidophylic fluorophore BTA-1. Arrows indicate dense cytoplasmatic inclusions. (b) mRFP intrinsic (red) fluorescence. (c) Staining with BTA-1 (green-blue). (d) Overlay of images (b) and (c), showing co-localization of the red and the greenish foci, which match with the inclusions in (a). Magnification bars: 2 µm.

Fig. 3 .

 3 Fig. 3. Dynamics of WH1(A31V)-mRFP inclusions in vivo, as followed by time-lapse microscopy. Two E. coli cells (labeled A and B) bearing inclusion particles are shown growing on LB-agar at 37ºC. Serial frames (odd rows, phase contrast; even rows, mRFP fluorescence) were taken at the indicated time intervals (lower right corner in the phase contrast images) starting 15 min after IPTG induction. Daughter cells are labeled as prime (first generation) and double prime (second generation). Arrows point to emergent fluorescent particles, colored according to their order of appearance (white yellow). Magnification bars: 2 µm.

Fig. 4 .

 4 Fig. 4. Ultrastructural analysis of amyloid inclusions in E. coli MC4100 cells. A. Confocal sections (∆z = 0.2036 µm) of bacteria expressing WH1(WT)-mRFP (left) or WH1(A31V)-mRFP (right). Rows: mRFP fluorescence (a, red); anti-His tag fluorescence (b, green); c, the overlay of (a) and (b). Arrows indicate amyloid primordia. Magnification bars: 3 µm. B. Immuno-electron microscopy of thin sections across bacteria expressing WH1-mRFP, either WT (top) or A31V (bottom). The boxed regions indicate the zoomed images to the right. Antibodies conjugated with colloidal gold spherical particles (ø ≈ 10 nm) decorate the primary antibody+His6(WH1) complexes, which are located on the electron-dense large protein aggregates in WH1(A31V), but are dispersed throughout the cytoplasm in WH1(WT). Arrow points to an amyloid primordium. Magnification bars: 200 nm.

Fig. 5 .

 5 Fig. 5. Purified WH1(A31V)-mRFP cellular inclusions seed the growth of amyloid fibers in vitro.A. Kinetics of amyloid growth, as followed by Th-T fluorescence emission. Pure soluble WH1 (WT or A31V; 35 µM) seeded (filled symbols) or not (empty symbols) with 1 µg of purified WH1(A31V)-

  B. TEM visualization of the protein aggregates obtained after four weeks incubation. Top right: standard WH1(A31V) amyloid fibers control grown in the presence of opsp-DNA. Magnification bars: 200 nm.

  ;

	physiologically relevant co-factors such as glycosamineglycans or phospholipids
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	8

Molecular Microbiology

Acknowledgements

We are grateful to Roger Y. Tsien for the gift of pRSETb-mCherry, to Ana M. Serrano, María T.

Seisdedos, Fernando González and Mercedes Carnota for technical assistance and to Salvador Ventura and Daniela Rhodes for the critical reading of this manuscript. This work was supported by grants from the Spanish Ministry of Science and Innovation (MICINN: BFU2006-00494, BIO2009-06952 and CSD2009-00088) and the Autonomous Government of Madrid (CAM: P-BIO-0214-2006).