extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Original Encounter with Antigen Determines Antigen-Presenting Cell Imprinting of the Quality of the Immune Response in Mice

Background Obtaining a certain multi-functionality of cellular immunity for the control of infectious diseases is a burning question in immunology and in vaccine design. Early events, including antigen shuttling to secondary lymphoid organs and recruitment of innate immune cells for adaptive immune response, determine host responsiveness to antigens. However, the sequence of these events and their impact on the quality of the immune response remain to be elucidated. Here, we chose to study Modified Vaccinia virus Ankara (MVA) which is now replacing live Smallpox vaccines and is proposed as an attenuated vector for vaccination strategies against infectious diseases. Methodology/Principal findings We analyzed in vivo mechanisms triggered following intradermal (i.d.) and intramuscular (i.m.) Modified Vaccinia virus Ankara (MVA) administration. We demonstrated significant differences in the antigen shuttling to lymphoid organs by macrophages (MΦs), myeloid dendritic cells (DCs), and neutrophils (PMNs). MVA i.d. administration resulted in better antigen distribution and more sustained antigen-presenting cells (APCs) recruitment into draining lymph nodes than with i.m. administration. These APCs, which comprise both DCs and MΦs, were differentially involved in T cell priming and shaped remarkably the quality of cytokine-producing virus-specific T cells according to the entry route of MVA. Conclusions/Significance This study improves our understanding of the mechanisms of antigen delivery and their consequences on the quality of immune responses and provides new insights for vaccine development.

Introduction
The in vivo mechanisms triggered by antigen delivery have been studied much less than those related to T and B-cell and protective immunity. In particular, the processes of local uptake and transport of antigen, the status of antigen-presenting cells (APCs), and the circumstances of T cell priming remain elusive.
Modified Vaccinia virus Ankara (MVA) is now replacing live Smallpox vaccines that were successfully used during decades without any knowledge of mechanism-inducing immune responses [1,2,3,4,5,6]. MVA is an attenuated strain of Vaccinia virus (VV) lacking the ability to replicate in vitro and in vivo [7,8,9,10] and is currently being investigated as a candidate recombinant vaccine for infectious diseases [11]. The efficacy of MVA immunization has been investigated in several animal models [1,3,4,6] however, without rigorous comparison of distinct routes of immunization. The use of conventional intramuscular (i.m.) and intradermal (i.d.) delivery routes for immunization remains part of the intense debate. We investigated whether the site of antigen delivery may dictate the differential participation of APCs in the initial steps of T cell priming, activation and quality of cellular immune responses. Deciphering the mechanisms involved in T cell mediated immunity is of particular interest for MVA, a leading option for the development of HIV vaccines and tumor immunotherapy [12].
The complex relationships linking APC identity, recruitment, antigen-loading, and localization, to the intensity and quality of immune responses make it necessary to analyze the early steps of T cell priming in greater detail. Human studies have highlighted the role of IFN-c-producing CD4 Th1 cells in the generation of longterm cell memory to VV [13,14], for roughly half of long-term vaccinees specifically lose their VV-specific CD8 T cell response [14]. In addition, recent studies in humans vaccinated with VV showed a major amplification of CD8 cells at priming [15]. These studies have focused on the magnitude of the effector T cell response, while recent works have underlined the importance of defining distinct functional T cell populations that reflect the quality of the T cell response [16]. Route-dependent effects of MVA have been investigated for the localization of CD8 T cell priming [17] and for antigen processing and MHC-Class I presentation [18]. It has also been shown that the immunization route may profoundly affect the recognition of MHC-Class I restricted poxvirus epitopes [19]. According to the literature, MVA has a pronounced tropism for monocytes (Mos)/macrophages (MWs) but also infects dendritic cells (DCs) and B cells, all of which can act as professional APCs [20,21]. In a model of VV subcutaneous (s.c.) administration, clustering was observed only between VV-specific CD8 + T cells and infected DCs, although most VV-infected cells in draining lymph nodes (DLNs) were MWs [22,23]. The direct involvement in T cell priming of other APCs, such as MWs, has so far not been demonstrated.
In this study, we investigated the early mechanisms of MVAinduced immune response after i.d. and i.m. immunization. Our findings demonstrate that i.d. and i.m. administration target different APCs that differentially shape the virus-specific cell-mediated immune response of both CD4 and CD8 cells. The accessibility of MVA antigen to different APCs at the site of immunization dictates the occurrence and extent of cellular immunity.

Results
The site of antigen delivery shapes both the intensity and the quality of MVA-induced cellular immune response Despite numerous comparisons of antigen delivery routes for the induction of immune responses against VV, there appears to be no comparisons between the standard i.d. and i.m. routes of immunization. We thus compared both the intensity and quality of cellular ( Figure 1) and humoral ( Figure S1) immune responses after MVA immunization by these routes. MVA-specific T cell responses in LNs draining the muscle (the inguinal DLNs, IDLNs) was much lower than the MVA-specific T cell response in LNs draining the skin (auricular draining lymph nodes, ADLNs) as assessed by an IFN-c production ELISPOT ( Figure 1A). Similar results were observed with MVA encoding for HIV-1-reverse transcriptase (rMVA-RT) ( Figure S2).
To determine whether the MVA delivery route induced distinct polyfunctional effector T cells, DLN cells were harvested seven days after MVA delivery at the peak of effector T cell responses ( Figure  S3). Simultaneous expression of IFN-c, TNF-a, and IL-2 by MVAspecific T cells was analyzed with flow cytometry and the Boolean gating function of FlowJo software ( Figure 1B-1E, and Figure S4). Of the various T cell functions, secretion of IFN-c, TNF-a, and/or IL-2 is particularly relevant for virus-specific T cell response studies [16]. As expected, the frequency of total cytokine-producing CD4 + T cells was significantly higher in the ADLNs of mice that received MVA i.d. than in IDLNs from mice immunized by the i.m. route ( Figure 1B). However, the frequency of cytokine-producing CD8 + T cells was similar in both groups, regardless of the route of inoculation ( Figure 1D). Remarkably, we noted significant differences in the multifunctionality of these induced T cells according to the site of antigen entry ( Figure 1C and 1E). Pie charts analyses summarize differences in quality of cytokine producing CD4 and CD8 cells after i.d. and i.m. immunization. In the group with MVA administered i.d., the MVA-specific CD4 + T cells producing TNFa were significantly higher compared to the MVA administered i.m. group ( Figure 1C). Strikingly, the total cytokine response of CD8 + T cells was sharply different according to the MVA administration route. The frequency of multiple cytokine-producing CD8 + T cells was higher after i.d. administration, and IFN-c-TNF-a production dominated the response ( Figure 1E).
The magnitude of MVA-specific T cell response was also higher in the spleen of mice that received MVA i.d. compared to those administered i.m., and differences in the multifunctionality of MVA-specific T cells were observed according to the site of antigen entry ( Figure S4).
Thus, it is not only the magnitude of the T cell response that is affected but also its quality: the MVA delivery route determines distinct patterns of cytokine production by antigen-specific T cells.
The persistence of MVA at the inoculation site induced prolonged and distinct innate cell recruitment The intrinsic structure of skin and muscle and the difference in their ability to be infiltrated by innate immune cells may modulate antigen persistence and its conveyance to lymphoid organs. To determine if the discrepancy observed between antigen-specific T cell responses resulted directly from early innate events at the antigen delivery site, we next evaluated the distribution of MVA at the inoculation sites and subsequent innate cell recruitment after i.d. and i.m. inoculation of MVA recombinant for the enhanced green fluorescent protein (rMVA-egfp) ( Figure 2).
As Figure 2A shows, localization of rMVA-egfp at inoculation sites was assessed at several time points using a fibered-confocal microscope (Cell VizioH, Mauna Kea technologies) [24]; high amounts of MVA particles were clearly present in the mouse skin (upper panels) and muscle (lower panels) and persisted there for 4 to 48 h after both i.d. and i.m. antigen delivery.
We then evaluated the nature, kinetics, and behavior of cells both resident in and infiltrating skin and muscle at several time points ( Figure 2B-2E). Analysis of cells isolated from the skin revealed a significant and sustained increase in CD11b hi CD11c 2 Ly-6G + Ly-6C + polymorphonuclear neutrophils (PMNs) in the dermis from 4 to 48 h after i.d. MVA inoculation ( Figure 2B, upper dot-plots, and Figure 2C). At the same time, the frequency of the subpopulation of CD11b + CD11c 2 F4/80 lo Mos increased strongly ( Figure 2B, lower dot-plots, and Figure 2C). In the dermis, two cell subsets expressing F4/80 were also identified as CD11b + F4/80 hi CD11c 2 dermal MWs and CD11b + F4/80 + CD11c + dermal DCs. The frequency of both fell at 4 and 48 h after i.d. MVA injection ( Figure 2B, lower dotplots, and Figure 2C).
After i.m. MVA delivery, however, we observed an early and transient increase in PMNs frequency at four h after inoculation that significantly fell at 48 h ( Figure 2D, upper panels, and Figure 2E). In addition, no significant changes in CD11c + DC frequency were observed. In contrast, the CD11c 2 F4/80 hi MW subset decreased slightly four h after i.m. antigen delivery and then increased continuously up to 48 h. This mirrored the behavior of the CD11c 2 F4/80 + Mo subset that declined at 48 h in the muscle. Because both Mo/MW subsets expressed high levels of Ly-6C and CD11b, the variation observed may represent phenotypic changes due to Mos differentiation rather than renewal during cell infiltration of muscle tissue.
Collectively, these results suggest that the dynamics and kinetics of innate cell populations differs significantly in skin and muscle. In particular, although the initial frequencies of Mos and MWs were similar at both inoculation sites, there were more DCs in the skin between 4 to 48 hours. The availability of APCs (i.e., DCs and MWs) in the skin may be a critical factor in shuttling antigen to lymphoid tissues and in its subsequent presentation to T lymphocytes.

Influence of the site of antigen delivery on the rate of antigen distribution toward DLNs
We further assessed the kinetics of the distribution of rMVA-egfp in DLNs and quantified the eGFP + cells by flow cytometry ( Figure 3A). The number of GFP-expressing cells peaked in ADLNs (after i.d. immunization) as early as four h after injection, at <4000 per ADLN ( Figure 3A). This number then declined rapidly with only <300 positive cells detected at 24 h and 48 h after i.d. inoculation of rMVA-egfp. eGFP + particles were detected in 72.568.6% of MWs (CD11b + F4/80 hi CD11c 2 ), in 1667.3% of myeloid DCs (mDCs, CD11c + CD11b + ) and 7.364.3% of PMNs ( Figure 3B). These results show that rMVA-egfp particles were associated with myeloid-derived cells in ADLNs within the first four h of i.d. antigen delivery. Extremely low amounts of GFP-expressing cells were observed in the IDLNs after i.m. injection ( Figure 3A); there were around 200 eGFP + cells at four h afterwards. This paucity of GFP-expressing cells prevented us from performing further analysis after i.m. injection.
We further assessed the presence of viral antigens in DLNs, using an anti-vaccinia antibody for histological analyses ( Figure 3C). After i.d. injection, MVA proteins were detected as early as four h afterwards in the ADLN subcapsular sinus, but also at 24 and 48 h in deeper areas beneath and between B cell follicles ( Figure 3C, upper panels). In contrast, after i.m. injection, low  Our results show for the first time major differences between the i.d. and i.m. routes for innate cell infiltration, virus shuttling, and antigenic distribution. Whereas i.d. inoculation leads to rapid transfer of MVA into the ADLNs with viral antigen persisting for at least the first 48 h after immunization, i.m. inoculation of MVA induces only slight dissemination of MVA particles at late time points.

Influence of the site of antigen delivery on the LN cellular microenvironment and subsequent cell recruitment
We next investigated cellular heterogeneity of innate cell recruitment and DC populations in DLNs where immune response begins. Indeed, others have shown that the accumulation of APCs after initial antigen encounter might be linked with the initiation of adaptive immune responses [25,26]. At several time points after i.d. and i.m. antigen delivery, DLNs were isolated for flow cytometric identification of cell subpopulations, including Mos, MWs, PMNs, and DCs ( Figure 4A-4E). The PMN subset multiplied by 13 in the ADLNs within four h of the i.d. MVA inoculation, compared with control mice, and remained three to four times higher at 24 and 48 h ( Figure 4A). In contrast, after i.m. immunization, PMN levels in IDLNs did not change significantly ( Figure 4A). This sharp contrast suggests differences in initial inflammation by i.d. and i.m. routes. In addition, increased amounts of CD11b + F4/80 hi MWs were detected at 24 and 48 h after i.d. but not i.m. inoculation ( Figure 4B). In contrast, CD11b + CD11c + mDCs quantitatively increased from 24 to 48 h after both i.d. and i.m. injections compared with their respective control mice ( Figure 4C). In addition, the number of CD11c + CD8 + lymphoid DCs remained constant both in ADLNs and IDLNs ( Figure 4D) and CD11c + B220 + plasmacytoid DCs (pDCs) increased at 24 and 48 h after MVA inoculation by both routes ( Figure 4E).
Thus, both i.d. and i.m. antigen delivery produced remarkable changes in myeloid and innate cell populations in the respective DLN suggesting that the initial influxes of leukocytes in DLNs may play a role in the intensity and quality of adaptive immune response to MVA antigens.

Differential APC involvement in T cell priming according to the original antigen delivery site
Because lymphoid organs are settled by successive and different waves of innate cells likely to be involved in antigen capture and presentation to T lymphocytes, we examined whether the accessibility of MVA antigen to different APC subpopulations dictates the occurrence and extent of cellular immunity. We isolated CD11c + DCs and CD11c 2 F480 + MWs ( Figure S5A) and tested their ability to stimulate IFN-c production by naive CD4 and CD8 T lymphocytes in a 36-hour IFNc-ELISPOT assay ( Figure 5). First, both DCs and MWs isolated from ADLNs after i.d. immunization induced much stronger MVA-specific effector CD4 + and CD8 + T cells than did those isolated from IDLNs after i.m. immunization ( Figure 5A) confirming superior intensity of cellular responses after i.d. immunization. The capacity of these  Figure 5B).
Because lymphoid and myeloid DCs have been a matter of debate in T cell priming, we next purified CD11b + CD11c + myeloid and CD8a + CD11c + lymphoid DCs to test their ability to prime T cells. Surprisingly, mDCs induced IFN-c production by CD4 + and CD8 + T cells more potently than lymphoid DCs ( Figure 5C).
These data demonstrate that APCs of myeloid origin are involved in T cell priming after MVA inoculation. i.d. antigen delivery mobilizes two distinct APC subsets, both mDCs and MWs, whereas i.m. inoculation appears to involve only the former.

Intradermal antigen delivery-dependent myeloid APCs imprinting of MVA-specific T cell responses
Although the main function of APCs is to trigger T cells into cell cycle progression, it is now well established that functionally distinct APC subsets can also influence the subsequent development of these dividing cells and their aptitude to secrete different patterns of cytokines [27]. We next tested whether the quality of APCs can directly influence the quality of the T cell responses. We found in our model that the most powerful DCs that stimulate T cells were also positive for the Ly-6C and F4/80 markers and expressed MHC II strongly ( Figure S5B). Purified F4/80 + cells (mDCs and MWs) isolated from control or i.d. MVA injected mice at day 2 post-immunization were then i.v. injected into recipient mice that had received MVA by the i.m. route ( Figure 6A). We analyzed the intensity of T cell effector immune response by measuring IFN-c production and we characterized the cytokine production patterns on day 7, by the multiparametric flow cytometric analysis in different groups described in Figure 6.
Interestingly, transferring F4/80 + cells isolated from i.d. injected mice, but not naive mice, increased the magnitude of MVAspecific T cell response ( Figure 6B), and shaped the cytokine distribution with increased frequency of TNF-a2producing CD4 + T cells ( Figure 6C, upper panel). Remarkably, the transfer of F4/ 80 + cells isolated from i.d. MVA injected mice, but not naive mice, to MVA i.m. inoculated mice restored the cytokine distribution of CD8 + T cells observed for the i.d. route ( Figure 6C, lower panel). The analysis of the splenic compartment led to similar observations ( Figure S6). Therefore, these results indicate that specific APCs originally imprinted at the site of immunization have the capacity to shape the quality of the immune response.

Discussion
The results presented in this report provide for the first time a demonstration of the impact of the antigen delivery route on the quality of the immune response, a quality that reflects the functional status of effector T cells. Several studies have emphasized the influence of environmental cues, antigen dose, and specificity of the APCs recruited to the T cell activation site in shaping the response by modulating cell function (reviewed in [27]). Here, we provide in vivo evidence that the route of delivery affects all these factors that determine the type of immune response. In particular, we demonstrated that i.d. injection resulted in better antigen distribution and more sustained APC recruitment into DLNs than with i.m. injection. These APCs, which comprise both DCs and MWs, are involved in T cell priming and are responsible for tuning the quality of the elicited immune response.
Many routes of immunization have been used to study poxvirusinduced immune responses in mice. However, we chose to compare the i.m. route, currently used in human vaccination trials to overcome untoward effects such as local skin reactions induced by s.c. or i.d. vaccine delivery, with the i.d. route, which closely mimics the original VV vaccination route of scarification. Strikingly, the mode of antigen delivery affected not only the strength but also the quality of the antigen-specific response, as measured by the total cytokine response.
The capacity of a viral vector such as MVA to elicit highfrequency immune responses depends critically on the efficient presentation of antigens to T lymphocytes. This may be particularly true for MVA, which has impaired replication ability. This observation led us to explore the capacity of the antigen to reach the secondary lymphoid organs and the identity of the immunocompetent cells recruited and potentially involved in T cell priming and subsequent T cell activation. Our data demonstrate that i.d. antigen delivery results in the rapid spread of the entire virus to DLNs in contrast to the i.m. antigen administration. The amount of antigen that reaches the DLN seems to be higher after i.d. compared to i.m. route, which could explain the intensity of the immune response of i.d. route compared to i.m. route. However both routes of immunization induced significantly intense immune responses, compared to control mice. Anatomic constraints may promote or prevent the dissemination of peripheral antigenic compounds, for the vasculature in muscle is much less extensive that than in skin, which has a sprawling network of lymphatic vessels. After i.m. inoculation, we found GFP associated mainly with muscle fibers. This is consistent with recent observations showing that after i.m. inoculation of a recombinant strain of MVA expressing the luciferase reporter gene, the luciferase signal was restricted mainly to the muscle [28]. Alternatively, the discrepancy in antigen transport from the site of inoculation toward secondary lymphoid organs may reveal differences in the capacity of innate immune cells to be recruited within the tissue and subsequently migrate towards these organs. This may result in differential inflammatory environments.
We also observed sharply different behavior of incoming leukocytes according to whether the inoculation site was skin or muscle. After i.d. inoculation, a sustained increase in the number of PMNs and Mos recovered from the skin was observed, while the increase in PMNs in the muscle after i.m. inoculation was only transient. Interestingly, at early time points after i.d. administration, MVA moved to the ADLNs at the same time as a substantial influx of both PMNs and Mos/MWs was also observed there. In view of previous studies demonstrating the role of Mos/MWs and PMNs in bacteria transport from the periphery to DLNs [29,30], the early burst of these cell populations at both the i.d. inoculation site and in the subcapsular sinus of DLNs suggests that these cells may have some role in shuttling the antigen. However, it is also possible that most of the viral particles may drain directly from the skin to ADLNs. In contrast, after i.m. antigen delivery, antigen positive cells were detected later into the deeper paracortex of DLNs.
MVA particles in ADLNs were found to be associated mainly with several cell populations of myeloid origin, specifically MWs, mDCs, and PMNs. Despite its non-replicating and non-propagating properties, MVA presents the same pattern of in vivo viral tropism as previously observed for s.c. injected VV [22]. In vivo MVA tropism and, in particular, its capacity to infect MWs and DCs is of crucial importance since both these cells function as professional APCs and could be involved in antigen presentation and in subsequent T cell priming.
Among APC subsets, MWs have been somewhat neglected because few reports produce evidence that these particular cells have any decisive function besides their phagocytic activity. While an immunostimulatory role in the generation of cytotoxic T lymphocytes was observed, their exact contribution to this antiviral activity remains unknown [31]. Intriguingly, we report here that Mos/MWs flowed massively into ADLNs after MVA i.d. inoculation. Previous work has noted the massive presence of VV-infected MWs in DLNs, but has pointed out only the role of infected DCs in direct VV antigen presentation to CD8 + T cells [22,23]. Our data demonstrate that MWs have a previously neglected role -the triggering of T cell priming after i.d. antigen delivery. Although less effective than DCs, MWs are capable of activating naive T lymphocytes [32]. Here, the contribution of MWs in T cell priming after MVA i.d. inoculation may be related to their local abundance in DLNs and the inflammatory environment [25,26]. We did not directly examine whether the differences we saw in the immunization routes was caused by direct or cross-presentation. Due to its ability to infect and to efficiently produce viral and recombinant antigens in both professional APC and non-professional APC, MVA has characteristics that enable direct priming as well as cross priming. A previous report suggested that the MVA-specific CD8 T cell response strongly though not exclusively depends on cross priming [33]. As the IFN-c RT response as a proportion of the overall MVA response ( Figure S2) was lower, it suggests that the increased overall MVA response may be due to cross-priming.
Both dermal DCs and CD8a + DCs are widely involved in the presentation of viral antigens [34,35,36,37,38,39]. In VV infection, the conventional CD8a + DCs are the main DC subset that primes the anti-VV CTL response in vivo [40]. Recent findings also reveal that pDCs can present VV antigens to CD8-T cells [41]. Unfortunately, because we recovered only small quantities of these pDCs from DLNs, we could not directly test them as APCs in our system. It is tempting, however, to hypothesize that they may have a decisive function, either directly by antigen presentation, or indirectly, by promoting other DCs as suggested by others [42]. Surprisingly, we found that among the DC subsets we tested, only mDCs rather than lymphoid DC efficiently presented MVA antigens to both CD4 + and CD8 + T cells. Nevertheless, the divergence between our results and those previously published may be due to the experimental system studying polyclonal antigen-responses to MVA. Indeed, our study examined the overall T cell response elicited in vivo, whereas mostif not all-previous data come from studies under optimal conditions in a peptide-specific transgenic T cell system and focused on class I-restricted presentation of viral antigens by DCs [22,23,40]. Taken together, these results challenge the idea that not only CD8a + DCs play an important part in priming immunity during poxvirus infection [22,40] but also mDCs could have a crucial role. However, MWs involvement in T cell priming would depend on the original site of antigen encounter.
Whether the mDCs able to present antigens arise from the peripheral tissues where they have phagocytosed MVA in situ remains to be confirmed. The phenotypes of dermis-and muscleinfiltrating cells as well as the phenotype of CD11b + CD11c + DCs recovered from ADLNs and IDLNs coincide with that of dermal DCs or DCs differentiated from Mos recruited to the inoculation site [36,43]. These CD11b + CD11c + DCs began increasing in frequency and number at 24 hr after antigen delivery and may therefore correspond to migratory DCs that have traveled from the periphery towards DLNs [36]. These results are in accordance with one antigen presentation scenario proposed by Villadangos and Schnorrer, involving both migratory and resident DC populations. In this scenario, the migratory DCs are themselves infected by the virus, which has no cytopathic effect, and thus present antigens by MHC class I and class II molecules in the DLNs [44]. This scenario is consistent with the phenotype of APCs involved in T cell priming in our model.
The generation of multifunctional CD8 + T cells with enhanced effector functions has been demonstrated after MVA vaccination in humans [45]. Among the functions tested, MVA induced mainly polyfunctional CD8 + T cells that produced MIP-1b, IFNc, and TNF-a. In our mouse model, the relative proportion of multiple cytokine production by CD8 cells was much higher in the i.d. (30%) than the i.m. (13%) group (p,0.05), whereas similar relative proportions of multiple cytokine-producing CD4 cells were found by both routes. After i.d. antigen delivery, single producers of IFN-c and TNF-a as well as CD8 + T cells producing both cytokines were dominant, whereas after i.m. administration, we detected mainly CD8 + T cells producing a single cytokine. The induction of multifunctional antigen-specific CD8 + T cells, in particular those producing both IFN-c and TNF-a, may contribute to effective immunity, for T cells with this functional signature persist in individuals vaccinated with VV [46]. This suggests that i.d. antigen delivery might be a better route for the generation and maintenance of multifunctional CD8 T cells.
An important finding is that F4/80 + cells isolated from ADLNs are endowed with the potential to modulate the cytokine production of T cells in i.m.-inoculated mice. Antigen presentation by distinct APCs results in different patterns of cytokine production in responding T cells [47]. The involvement of MWs in T cell priming after i.d. antigen delivery may therefore account for the effect on the immune response of this MVA administration route. Together, these results suggest that the quality of antigenspecific T cells is imprinted early after antigen encounter and might reflect differences in cell tropism, antigen accessibility and presentation.
Overall, these results emphasize the contribution of migratory cells to MVA-induced T cell priming and suggest that the in vivo antigen distribution, the mobilization of particular APC subsets toward the DLNs, and their anatomical location may together explain the differences we observed in MVA-elicited immune responses according to the delivery route. We have demonstrated that this antigen delivery route is of crucial importance in driving immunocompetent cells to lymphoid organs where they subsequently induce distinct functional populations of T cells. This study improves our understanding of the mechanisms of antigen delivery and may provide new insights for vaccine development.

Mice and immunizations
Female C57BL/6 mice (6-8 weeks old) were obtained from Charles River Laboratories (L'Arbresle, France) and were housed under specific pathogen-free conditions. All animals were handled in strict accordance with good animal practice and complied with local animal experimentation and ethics committee guidelines.

Interferon-c ELISPOT assay
The IFN-c ELISPOT kit (Diaclone, Strasbourg, France) was used under the manufacturer's suggested conditions. Briefly, DLN cells (1.0610 5 cells) were harvested at day 7 after MVA immunization. Cells were incubated with MVA (0.1 PFU/cell) or overlapping HIV-1-RT 15 mer-peptides for 36 hr at 37uC on a plate coated with purified anti-IFN-c. After incubation, the plates were incubated with biotinylated anti-IFN-c mAb for 90 min., then by streptavidin-alkaline phosphatase conjugated for 1 hour and BCIP/NBT substrate buffer. The frequency of spot-forming cells (SFC) was measured with an automated microscope (Zeiss).

MVA-egfp neutralization assay
Neutralizing activities were evaluated by an assay based on flow cytometric detection of GFP as previously described [48]. A detailed description of the method used is included Figure S1.

Preparation of cell suspensions
Ears, muscles, and lymph nodes (LN) cells were dissociated from the matrix by a 45-minute incubation at 37uC with 400 units/ml collagenase (Sigma-Aldrich) in RPMI 1640 medium (Invitrogen). Cells were then filtered through a 70-mm nylon mesh, and counted before Ab staining.

Intracellular cytokine staining
For intracytoplasmic cytokine detection, cell suspensions were stimulated overnight with MVA (0,1 PFU/cell) and then incubated for 4 hr at 37uC with brefeldin A at 5 mg/ml. The cells were stained with CD4-PercP (clone RM4-5) or CD8a-PercP (clone 53-6.7). After fixation and permeabilization with 1X PBS-2% FCS-0.1% saponin, cells were stained with APC-conjugated anti-mouse IFN-c, FITC-conjugated anti-IL-2, and PE-conjugated anti-TNFa (BD Biosciences). Cells were then run on a FACSCalibur (BD Biosciences, San Jose, CA): 50,000 live CD8 or CD4 positive events per sample were analyzed with FlowJo software (Treestar, Inc., San Carlos, CA). Boolean combination gating was performed to determine the frequencies of expression profiles corresponding to the seven different combinations of cytokines. For ex-vivo APC assays, nitrocellulose 96-well plates (Millipore) were coated as described above. MACS-isolated CD4 + or CD8 + T cells obtained from naive LNs were plated at 10 6 cells/well. The indicated numbers of sorted DC populations (total DCs, MWs, myeloid DCs, CD8AE + DCs) from DLNs on day 2 after i.d. and i.m. MVA inoculation were added to the wells in 100-ml aliquots. After 36 hr of incubation at 37uC, the plates were treated and SFC frequencies determined as described above.

Isolation of DCs and MWs from lymph nodes and activation of naive T cells
Adoptive transfer of F4/80 + cells DLNs taken on day 2 from ten mice inoculated i.d. with MVA or from control mice were treated with collagenase, filtered through a 70-mm nylon mesh and layered on a Histodenz gradient. After low-density cells were collected and washed in PBS-FCS, F4/80 + cells were isolated by using F4/80-PE and anti-PE coated magnetic microbeads (MACs, Miltenyi Biotec). 5.10 5 F4/80 positive cells were injected i.v. into i.m.-MVA inoculated mice on day 2. At day 7, the DLN T cell cytokine profile was determined by intracellular cytokine staining.

In vivo confocal microscopy
In vivo biodistribution of rMVA-egfp was monitored with noninvasive fiber-based confocal microscopy (CellVizioH, Mauna Kea Technologies) as previously described [24]. The S-1500 optical probe used for acquisition of the images presented here has a diameter of 1.5 mm providing images immediately below the surface of biological tissue, with a slice thickness of 15 mm and a lateral resolution of 5 mm. Fluorescent spots in 10 different areas per organ were collected for at least 3 mice (n = 30). Saline buffertreated mouse tissue was used for background fluorescence detection.

Immunofluorescence on tissue sections
Sections cut from frozen DLNs were incubated with rat anti-CD45R/B220, biotinylated, rabbit-anti-vaccinia (AbCys s.a.), purified rat anti-F4/80, or purified hamster anti-CD3e antibodies. Then, slides were incubated with secondary antibody goat anti-rat IgG Alexa 488, or 633, anti-hamster-Alexa 594, Alexa FluorH488 or Texas red conjugated-streptavidin (Probes, Invitrogen). Slides were mounted with Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL) and analyzed under an Olympus fluorescence microscope. Images were acquired with a Evolution VF/Qimaging digital camera and Qimaging QCapture Pro software. Images were then processed with Adobe Photoshop (Adobe Systems, San Jose, CA). For confocal microscopy, we used the Leica Sp2 AOBS confocal microscope and processed the images with Leica imagej64 software.

Statistical analysis
Data are presented as the mean6standard deviation. Statistical analyses compared the groups of mice with the unpaired t-test and Graphpad software. P values of less than 0.05 were considered statistically significant. Figure S1 Time course of antibody response to MVA immunization. Groups of mice (six per group) were immunized i.d. (filled circle) or i.m. (filled square) with 5.106 PFU of MVA. Sera were collected at different time points following immunization and neutralizing antibody titers were determined using the neutralization assay that measures the reduction in infectivity of rMVA-egfp. This assay was performed by adding 2.5610 4 PFU of rMVA-egfp to 40 ml of serial dilution of heat-inactivated serum. The plate was incubated for 1 hr at 37uC. Then, 1610 5 HeLa cells were added to 50 ml of culture and incubated for 16 hr at 37uC. GFP expression was analyzed on a total of 10,000 live events per sample with a FACSCalibur and CellQuestPro software (BD Biosciences). The percentage of neutralization was defined as ratio of the reduction in the number of GFP-expressing cells to the number of GFP-expressing cells in untreated control wells and calculated as follows: (12[percentage of GFP-expressing cells/ percentage of GFP-expressing cells in untreated controls])6100. The serum dilution that reduced the percentage of GFPexpressing cells by 50% (IC50) was determined by nonlinear regression with the PRISM software package (version 4.00; GraphPad Software, Inc., San Diego, CA). Geometric mean titres are shown. Differences between i.d. and i.m. immunized groups were analyzed using unpaired t-test, **P,0.01. or i.m. with 5.106 PFU of rMVA-RT or saline buffer as a control. Seven days following vaccination, mice were killed, and DLNs were harvested, and stimulated with overlapping HIV-1-RT 15 mers peptides in vitro. IFN-c-producing T cells were evaluated by ELISPOT assay. Representative data from two independent experiments are presented as the mean6standard deviation (n = 11 individual mice). * (P,0.05) represents the differences between mock-injected mice and MVA-inoculated mice, (NS, Non Significant). The statistical differences values between i.d. and i.m.-MVA inoculated groups of mice are indicated. Found at: doi:10.1371/journal.pone.0008159.s002 (1.85 MB TIF) Figure S3 Time course of MVA-specific T cell response in the DLNs. Groups of mice were immunized i.d. or i.m. with 5.106 pfu of MVA or saline buffer as a control. Seven, fourteen, thirty, and sixty days following vaccination, mice were killed, and DLNs were harvested. IFN-c-producing T cells were evaluated by ELISPOT assay. Representative data from two (days 30 and 60) or three (days 7 and 14) independent experiments are presented as the mean6standard deviation (n = 16 individual mice for day 7 and day 14, n = 6 individual mice for day 30 and 60). ** (P,0.01) *** (P,0.001) represent the differences between between i.d. and i.m. MVA-immunized groups.


s2fieldsofstudy: Biology Chemistry

text:
Phosphoregulation of the budding yeast EB1 homologue Bim1p by Aurora/Ipl1p

EB1 (end binding 1) proteins have emerged as central regulators of microtubule (MT) plus ends in all eukaryotes, but molecular mechanisms controlling the activity of these proteins are poorly understood. In this study, we show that the budding yeast EB1 protein Bim1p is regulated by Aurora B/Ipl1p-mediated multisite phosphorylation. Bim1p forms a stable complex with Ipl1p and is phosphorylated on a cluster of six Ser residues in the flexible linker connecting the calponin homology (CH) and EB1 domains. Using reconstitution of plus end tracking in vitro and total internal reflection fluorescence microscopy, we show that dimerization of Bim1p and the presence of the linker domain are both required for efficient tip tracking and that linker phosphorylation removes Bim1p from static and dynamic MTs. Bim1 phosphorylation occurs during anaphase in vivo, and it is required for normal spindle elongation kinetics and an efficient disassembly of the spindle midzone. Our results define a mechanism for the use and regulation of CH domains in an EB1 protein.

Introduction
Microtubules (MTs) are highly dynamic polymers formed by a cylindrical assembly of -tubulin subunits that have essential functions for intracellular transport, chromosome segregation, and cellular architecture. Several MT-associated proteins regulate their dynamics and interactions and help to organize the MT cytoskeleton. An important subgroup among MT-associated proteins is constituted by the plus end-tracking proteins (+TIPs), which are characterized by their preferential association with growing MT plus ends in vivo. +TIPs are highly conserved among all eukaryotes and they can be divided into different subfamilies, including the DIS/chTOG family of XMAP215-like proteins, the CLIP-170 or CLASP-related families, spectraplakins, and, finally, the EB1 (end binding 1) family of MT endbinding proteins (for reviews see Akhmanova and Hoogenraad, 2005;Akhmanova and Steinmetz, 2008). EB1 proteins have emerged as key regulators of MT plus ends in all eukaryotes (Vaughan, 2005). They contain an N-terminal calponin homology (CH) domain, which displays MT-binding activity and is part of various tubulin-or actin-binding molecules (Hayashi and Ikura, 2003;Slep and Vale, 2007). The following C-terminal coiled-coil mediates homodimerization and is followed by a four-helix bundle (EB1 homology domain), which is critical for the tethering of cargo proteins to EB1 (Honnappa et al., 2005;Slep et al., 2005). Although crystal structures of the individual CH domain and the C-terminal cargo-binding domain have been solved, the spatial organization of these domains in the context of a full-length EB1 molecule remains unknown. Two recent studies of the Schizosaccharomyces pombe EB1 homologue Mal3 have provided some basic insights into the function of EB1 proteins. Using an EM approach, Sandblad et al. (2006) have investigated the decoration of stabilized MTs by Mal3 and found that the protein preferentially associates with the lattice seam of the MTs, the structurally most labile part of the polymer. Thus, it may act as a "molecular zipper" supporting the stabilization of the closing lattice seam (Sandblad et al., 2006;Vitre et al., 2008). In a different approach, Bieling et al. (2007) have reconstituted the interaction of three S. pombe +TIPs with dynamic MTs in vitro. Notably, only the EB1 homologue Mal3p had an intrinsic ability to track the MT end and was sufficient to localize the kinesin Tea1p and the CLIP-170 protein Tip1p to growing ends in vitro (Bieling et al., 2007). Despite this progress, important questions concerning EB1-related proteins have remained unanswered. For example, what is the molecular mechanism by which these proteins bind to the MT lattice and to the growing end? Moreover, how does the cell E B1 (end binding 1) proteins have emerged as central regulators of microtubule (MT) plus ends in all eukaryotes, but molecular mechanisms controlling the activity of these proteins are poorly understood. In this study, we show that the budding yeast EB1 protein Bim1p is regulated by Aurora B/Ipl1p-mediated multisite phosphorylation. Bim1p forms a stable complex with Ipl1p and is phosphorylated on a cluster of six Ser residues in the flexible linker connecting the calponin homology (CH) and EB1 domains. Using reconstitution of plus end tracking in vitro and total internal reflection fluorescence microscopy, we show that dimerization of Bim1p and the presence of the linker domain are both required for efficient tip tracking and that linker phosphorylation removes Bim1p from static and dynamic MTs. Bim1 phosphorylation occurs during anaphase in vivo, and it is required for normal spindle elongation kinetics and an efficient disassembly of the spindle midzone. Our results define a mechanism for the use and regulation of CH domains in an EB1 protein.
well-conserved CH and EB1-like domains (Fig. 1 B). Closer inspection of the phosphorylation sites revealed that most of them contained a consensus sequence targeted by the Aurora B kinase (Cheeseman et al., 2002), suggesting that Bim1p may be an Ipl1p substrate. To determine whether Bim1p is indeed phosphorylated by Ipl1/Aurora B, we expressed and purified full-length Bim1p from Escherichia coli, phosphorylated the protein with bacterially expressed Ipl1p in complex with its activator protein Sli15p, and analyzed the products of the kinase reaction by mass spectrometry. We found that precisely the same six phosphorylation sites we mapped in vivo were phosphorylated by purified Ipl1-Sli15 complex in vitro ( Fig. 1 B). To further validate the phosphorylation sites, Bim1p variants in which the six mapped phosphorylation sites were either mutated to Ala to prevent phosphorylation (Bim1 6A ) or mutated to aspartate to mimic constitutive phosphorylation (Bim1 6D ) were expressed and purified from bacteria. Although wild-type (WT) Bim1p (Bim1p WT ) was an excellent substrate for Ipl1-Sli15 in vitro, changing the mapped phosphorylation sites to Ala or Asp prevented phosphorylation almost completely, demonstrating that the phosphorylation is highly specific for the mapped residues ( Fig. 1 C). We also compared the efficiency of Bim1p phosphorylation either by Ipl1p alone or by Ipl1p and Sli15p. Although phosphorylation of Bim1p by Ipl1 alone was barely detectable under standard kinase assay conditions, the addition of Sli15p led to rapid phosphorylation (Fig. S1). Thus, effective phosphorylation of Bim1p by Ipl1p depends on the presence of Sli15p. Collectively, these results establish that Bim1p is a target of Ipl1p and that phosphorylation occurs on a cluster of six Ser residues located in the flexible linker region.

Bim1p forms a stable complex with Ipl1p via its C-terminal domain
Because Bim1p is phosphorylated by Ipl1p, we next explored which protein segments are important for mediating kinasesubstrate interactions. A series of Bim1p N-and C-terminal truncation mutants were expressed, purified as GST fusion proteins, and tested for interaction with purified Ipl1-Sli15 complex in pull-down assays. We found that Ipl1-Sli15 could bind to Bim1p and that the interaction required the presence of the C-terminal residues 185-344 encompassing the EB1 homology domain, which is required for the binding of other cargo molecules to EB1 family members ( Fig. 1 D and Fig. S2). The association between Bim1p and the Ipl1 complex was not affected by the phosphorylation status of Bim1p, as the phosphomimicking Bim1 6D mutant could interact effectively with Ipl1-Sli15. Furthermore, we observed that Bim1p can interact with Ipl1p also in the absence of Sli15p (Fig. S2). The Bim1-Ipl1 interaction was very robust and could be detected by analytical size exclusion chromatography (SEC) under physiologically relevant salt concentrations of 100 mM KCl ( Fig. 1 E). Interestingly, upon Ipl1p binding, the elution position of Bim1p was shifted toward a smaller Stokes radius, suggesting that interaction with Ipl1p stabilized a more compact conformation of the Bim1 molecule. In further support of a direct association, a recent genome-wide two-hybrid analysis regulate EB1 activity to control the dynamics and transport of proteins to plus ends? Because EB1 proteins constitute the center of extensive +TIP networks and control the association of many proteins with plus ends, they are particularly important targets for regulatory mechanisms (Lansbergen and Akhmanova, 2006).
Plus ends establish the contact of MTs to various cellular structures, among them the kinetochore, a complex assembly of proteins on centromeric DNA which mediates the attachment of chromosomes to the mitotic and meiotic spindle Cheeseman and Desai, 2008;Tanaka and Desai, 2008). Regulation of kinetochore-MT attachments is particularly important in the process of biorientation, i.e., the pathway by which the cell ensures that duplicated sister chromatids are connected to opposite spindle poles (Tanaka et al., 2005). In addition, the MT plus ends of interpolar MTs are bundled to form the spindle midzone, which is required for proper spindle elongation (Khmelinskii et al., 2007) and cytokinesis (Norden et al., 2006). The mitotic kinase Aurora B is thought to be critical for the regulation of MT plus ends in both of these cellular situations. In the process of kinetochore biorientation, Aurora B phosphorylates kinetochore components to release improper attachments (Cheeseman et al., 2002;Ciferri et al., 2008), and during anaphase, Aurora B localizes to the spindle midzone, where it is involved in timely spindle disassembly (Buvelot et al., 2003). To understand how Aurora B performs its critical functions, it is essential to identify all relevant downstream targets and elucidate their mechanism of regulation.
In this study, we identify the budding yeast EB1 homologue Bim1p as an Ipl1 substrate and provide molecular insights into its regulation. Biochemical analysis has revealed that Bim1p directly associates with the Ipl1p kinase and, upon activation by Sli15p, becomes phosphorylated on a cluster of six sites in the flexible linker domain. Our analysis indicates that the Bim1p linker domain is critical for MT binding, autonomous plus end tracking, and phosphoregulation of this EB1 family member.

Bim1p is phosphorylated by the Ipl1-Sli15 complex at the linker domain
To analyze the protein-protein interactions and posttranslational modifications of the budding yeast EB1 homologue Bim1p, we constructed a strain in which the endogenous BIM1 gene was tagged at the C terminus with a tandem affinity tag. The protein was purified from yeast extracts of logarithmically growing cells, and the preparation was analyzed after in-solution digest by mass spectrometry (Fig. 1 A). Under our stringent purification conditions (300 mM KCl), the budding yeast adenomatous polyposis coli homologue Kar9p, a known interaction partner of Bim1p with a role in orienting the yeast spindle (Hwang et al., 2003), was copurified. In addition, we mapped a total of six Bim1 phosphorylation sites. Interestingly, the identified sites (Ser139, were not randomly distributed throughout the molecule but clustered in a region of 40 aa in the flexible linker region connecting the

Multisite linker phosphorylation reduces the affinity of Bim1p for MTs
As the principal function of EB1 proteins relies on their ability to bind MTs, we next analyzed how linker phosphorylation by the Ipl1-Sli15 complex would influence the MT-binding activity of Bim1p. We compared the binding affinities of Bim1p WT , Bim1p phosphorylated in vitro with the Ipl1-Sli15 complex, and the phosphomimicking Bim1 6D mutant for taxol-stabilized MTs in cosedimentation assays. Bim1p WT displayed a robust affinity for MTs with an apparent dissociation constant (K d ) of 0.44 µM. MT binding of Bim1p phosphorylated by Ipl1-Sli15 was markedly decreased, and the apparent K d was raised about fivefold. The phosphomimicking Bim1 6D mutant displayed an even lower affinity for taxolstabilized MTs (Fig. 3 A). We next asked whether Ipl1-Sli15 phosphorylation could also release Bim1p from the lattice after it had been prebound to taxol-stabilized MTs. We loaded MTs with Bim1p, incubated them with different amounts of purified Ipl1-Sli15 complex either in the absence or presence of ATP, and separated supernatant and pellet fractions after centrifugation. Only in the presence of ATP, Bim1p was removed by Ipl1-Sli15 from the pellet fraction into the supernatant ( Fig. 3 B). Thus, Ipl1-Sli15-mediated phosphorylation of the Bim1p linker domain decreases the affinity for taxolstabilized MTs in vitro and removes prebound Bim1p from the MT lattice.
To analyze the consequences of phosphorylation on Bim1p binding to MTs in vivo, we integrated Bim1 WT , Bim1 6A , and Bim1 6D variants fused with their C termini to 3× GFP into yeast. We expressed these Bim1p variants under their own promoter in a strain lacking endogenous BIM1 and visualized them by fluorescence live cell microscopy. Western blotting indicated that the Bim1 variants were expressed in similar amounts (Fig. 3 D). Bim1 WT -3× GFP showed a typical localization pattern with individual dots corresponding to MT plus ends and a prominent localization to the spindle midzone. The localization pattern of the 6A mutant appeared similar, although the spindle midzone seemed to be more prominently stained than in the WT control. However, the phosphomimicking 6D mutant displayed only very weak spindle staining, the cytoplasmic background signal was stronger, and only weakly fluorescent Bim1p dots remained visible ( Fig. 3 C), suggesting that constitutive phosphorylation of the linker domain also impairs binding of Bim1p to MTs in vivo.
We tested the effect of the multisite phosphorylated linker on the overall structure of Bim1p. We first compared the hydrodynamic properties of WT and phosphomimicking Bim1p proteins. Bim1p WT elutes remarkably early during SEC with a Stokes radius of 58 Å, suggesting a highly elongated shape of the dimeric molecule. However, the elution peak of the phosphomimicking Bim1 6D mutant was shifted to a later elution volume ( Fig. S3 A). This different migration behavior in SEC did not correspond to the presence of dimeric versus monomeric forms, as a Bim1p variant lacking the coiled-coil dimerization motif eluted later than the 6D mutant, reflecting its monomeric nature (Fig. S4). Thus, phosphorylation of the linker domain may lead to a conformational change of the dimeric Bim1 molecule, which results in a compaction. To further analyze the consequences of linker phosphorylation, we subjected Bim1 WT , Bim1 6A , and Bim1 6D variants to limited proteolysis using the protease chymotrypsin. Although WT and the 6A mutant showed similar degradation patterns, the digestion pattern for the 6D mutant was significantly altered, arguing for a profound conformational change exposing different cleavage sites ( Fig. S3 B). Thus, full-length Bim1p is a highly elongated molecule, and linker phosphorylation by the Ipl1-Sli15 complex may trigger a conformational change that results in a more compact dimeric molecule.

Bim1p phosphorylation occurs during anaphase in vivo
To study the phosphorylation status of Bim1p in vivo, we analyzed the pattern of epitope-tagged Bim1p by Western blotting. To enhance a potential phospho shift, SDS-PAGE was performed in the presence of the Phos-tag reagent (Kinoshita et al., 2006). In extracts derived from WT cells, Bim1p displayed slower migrating forms, which were eliminated after shifting the cells to the restrictive temperature in an ipl1-2 mutant (Fig. 2 A). To study the timing of Bim1 phosphorylation in vivo, we synchronized cells expressing Bim1-myc and the yeast securin Pds1-myc in G1 phase of the cell cycle with  factor, released the cells, and analyzed the phosphorylation pattern by Western blotting. Interestingly, Bim1 phosphorylation was detected in a narrow time window of 15-30 min after the beginning of Pds1 degradation (Fig. 2 B). Thus, Bim1 phosphorylation depends on Ipl1 and is detected specifically during anaphase in vivo. MTs prompted us to further investigate the mechanism of MT binding by Bim1p. To establish the molecular requirements for MT binding more rigorously, we constructed, expressed, and purified eight Bim1p variants encompassing different combinations of CH, linker, and dimerization domains as C-terminal EGFP fusion proteins (Fig. 4, A and B; and Fig. S5). This

CH and linker domains synergistically enhance MT binding of Bim1p
The MT-binding activity of EB1-like proteins had previously been assigned to the CH domain (Hayashi and Ikura, 2003). However, our surprising finding that phosphorylation of the linker region substantially influences the binding of Bim1p to (residues 1-344) bound to MTs with an apparent K d of 0.2 µM. In contrast, a monomeric construct encompassing the CH and the linker domain but lacking the coiled-coil dimerization motif (residues 1-210) showed a much lower affinity for allowed us to analyze the MT-binding activities in a fluorescencebased cosedimentation assay (see Materials and methods). We first tested the MT-binding activities of monomeric versus dimeric Bim1 variants (Fig. 4 C). Full-length Bim1-EGFP We first analyzed the behavior of full-length Bim1p WT in the in vitro tracking assay: 70 nM Bim1-EGFP was added together with free tubulin into the flow chamber. Two-color time-lapse imaging demonstrated that Bim1-EGFP preferentially accumulated at MT ends during phases of growth but was rapidly lost from the MT upon disassembly (Fig. 5 C and Videos 1 and 2). Although the majority of Bim1p accumulated at growing plus ends, there was always a significant amount of Bim1p also bound to the MT lattice, which increased upon lowering the ionic strength of the solution or raising the Bim1p concentration in the assay (unpublished data). In alternative experiments, we followed the plus end tracking with Bim1p covalently conjugated to Alexa Fluor fluorophores, either via Cys or amine linkages (unpublished data). We did not observe significant differences between the Alexa Fluor conjugates and the EGFP fusion protein. Thus, we conclude that the budding yeast EB1 homologue Bim1p autonomously tracks growing MT ends in vitro.
The molecular requirements for autonomous plus end tracking have not been established in a defined in vitro system. Therefore, we tested our engineered Bim1 constructs in the in vitro tracking assay and asked whether they could associate with the MT lattice or with growing ends (Fig. 5 B). Under conditions in which Bim1p WT displayed robust plus end tracking (70 nM final Bim1p concentration), the monomeric variant (Bim1 1-210 ) only weakly associated with dynamic MTs. Raising the final concentration of Bim1p 1-210 to 1 µM yielded a faint staining all along the MT lattice, whereas a substantial Bim1 accumulation at growing ends could not be observed (Fig. 5 C and Video 3). Thus, a monomeric Bim1p variant containing the CH and the linker domain failed to plus end track in vitro. In agreement with its low MT-binding affinity in the cosedimentation assay, the dimeric Bim1 variant lacking the linker domain (Bim1 1-133^210-344 ) was barely visible on MTs under our imaging conditions and did not display tip-tracking activity. Thus, dimerized CH domains and the presence of the linker are both necessary for the autonomous plus end-tracking activity of Bim1p.
To visualize the effect of Ipl1-Sli15 phosphorylation, we first analyzed the interaction of Bim1p with stable MT substrates. We incubated rhodamine-labeled, taxol-stabilized MTs with Bim1p-Alexa Fluor 488 and observed the binding reaction by TIRF microscopy. Bim1p-Alexa Fluor 488 decorated taxolstabilized MTs along their length without any observable preference for MT ends. We included purified Ipl1-Sli15 complex in the binding reaction, and during recording, we introduced ATP into the flow chamber. Within seconds after ATP addition but not after a buffer control, the Bim1p-Alexa Fluor 488 signal disappeared from taxol-stabilized MTs (Fig. 5 D and Video 4). Finally, we investigated the effect of Ipl1 phosphorylation on plus end tracking in vitro. We performed the tip-tracking assay as described in this section but now added purified Ipl1-Sli15 complex either in the presence or absence of ATP. After initial association with growing plus ends, the Bim1 signal disappeared from the plus ends as well as from the lattice. This was not the case in control assays in which either the Ipl1-Sli15 complex or ATP was omitted from the reaction (Fig. 5 E and Video 5). These results demonstrate that linker phosphorylation by Ipl1p removes Bim1p from dynamic MTs in vitro.
taxol-stabilized MTs, and the K d was raised nearly 20-fold to 3.5 µM. Multisite phosphorylation by Ipl1p further lowered the affinity of this construct for MTs to 4.8 µM (Bim1 6D 1-210 ). Finally, a construct containing only the CH domain fused to EGFP (Bim1 1-133 ) bound to MTs but only weakly, with an apparent K d of 8.9 µM.
In the next series of experiments, we compared the MTbinding activities of different dimeric Bim1 constructs (Fig. 4 D). Compared with full-length Bim1p, a variant lacking the CH domains (Bim1 134-344 ) displayed an 40-fold lower MT-binding activity. Multisite phosphorylation of the linker domain further reduced the MT affinity of this construct. Interestingly, a dimeric Bim1 variant that contained both CH domains but lacked the flexible linker residues (Bim1 1-133^210-344 ) displayed an MT-binding activity that was as low as the construct missing the CH domains.
We tested the effects of our engineered Bim1p constructs on tubulin polymerization using a light-scattering assay. As expected, full-length Bim1p strongly promoted MT assembly in vitro. Under the same experimental conditions, the phosphomimicking 6D mutant and the monomeric Bim1 variant containing the CH and linker domain both displayed a strongly reduced ability to promote tubulin polymerization. A construct encompassing the CH domain alone was completely ineffective in promoting polymerization (Fig. 4 E). To exclude the possibility that the observed effects are caused by the fusion with EGFP, we repeated these experiments with an additional set of constructs that contained EGFP connected by a C-terminal flexible 36-aa linker and did not see any significant changes in protein behavior (unpublished data).
Collectively, these results demonstrate that the CH domain of Bim1p is necessary but not sufficient for MT binding of the molecule. The flexible linker region contributes significantly to MT binding, and CH and linker domains synergistically allow MT binding of the full-length Bim1p molecule.

In vitro reconstitution of Bim1p plus end tracking and regulation
In the cell, Bim1p exerts its functions on dynamic MTs. Thus, we aimed to reconstitute and directly visualize the interaction of Bim1p with dynamic MTs in vitro. To this end, we modified a microscopy flow cell assay, which was previously used to analyze the dynamics of the Dam1 ring complex (Westermann et al., 2006). We adhered short, brightly labeled, biotinylated MT seeds that had been stabilized with the slowly hydrolyzing GTP analogue GMPCPP (guanosine 5-[,-methylene]triphosphate) to the surface of a coverslip via a biotin-streptavidin interaction (Fig. 5 A). After washing away any unbound seeds, dimly labeled free tubulin at a concentration of 15 µM was introduced. MTs were visualized at 30°C using total internal reflection fluorescence (TIRF) microscopy to eliminate the background fluorescence generated by unincorporated labeled tubulin dimers. Under these conditions, individual MTs grew from the stable seeds and displayed dynamic instability; i.e., phases of growth were followed by rapid disassembly before another growth phase started. A similar experimental setup has recently been used by Bieling et al. (2007) to investigate the properties of the S. pombe EB1 homologue Mal3p.

Functional organization of EB1 proteins
The molecular mechanisms regulating the activity of EB1 proteins have so far remained elusive. In this study, we identify the flexible linker region as the critical regulatory domain of the budding yeast EB1 homologue Bim1p (Fig. 7 A). This was unexpected, as previous work had assigned the MT-binding activity to the CH domain, and it has been speculated that the regulatory mechanisms may target this region of the molecule (Hayashi and Ikura, 2003). Among EB1 homologues, the sequence of the linker region is less well conserved than the CH and dimerization domains. However, the approximate length of the linker, its predicted unstructured nature, and its overall basic electrostatic character are common features of all EB1 proteins. A recent study suggests that the linker region may also be of critical importance for the S. pombe EB1 homologue Mal3, as C-terminal truncations of a monomeric Mal3 construct abolished its polymerization-promoting activity (des Georges et al., 2008). There is further evidence that the observed interaction between Bim1p and Ipl1p may be evolutionary conserved: using two-hybrid and coimmunoprecipitation analyses, a recent study identifies human Aurora B as an interaction partner of EB1 (Sun et al., 2008). Although a direct phosphorylation of EB1 by Aurora B could not be detected in vitro, which may be caused by the absence of an activator protein, there remains the possibility that either EB1 or the related proteins EB2 and -3 may be relevant physiological Aurora B targets. Furthermore, we did not obtain experimental evidence that Bim1p is regulated by autoinhibition between the CH domains and the extreme C termini, as has been proposed for EB1 (Hayashi et al., 2005). The reason may be that Bim1p is significantly larger than EB1 (344 residues compared with 268 for EB1) or that the C-terminal residues (ETF for Bim1p) differ from the sequence found in EB1 (EEY).

MT-binding interfaces
With the identification of Bim1p as an Ipl1 target, it is noteworthy to point out striking similarities between its regulation of MT binding and that of the Ndc80 kinetochore complex. Both molecules exploit CH domains as part of their MT-binding interfaces (Cheeseman et al., 2006;Wei et al., 2007), yet in both cases, unstructured regions located adjacent to the globular CH domain contribute significantly to MT binding and are the target of Aurora B phosphorylation at a cluster of neighboring residues (Ciferri et al., 2008;Guimaraes et al., 2008;Miller et al., 2008). Recent cryo-EM studies describing the decoration of MTs by Ndc80/Nuf2 (Wilson-Kubalek et al., 2008) or by the S. pombe EB1 homologue Mal3 (des Georges et al., 2008) suggest that the CH domain nestles into a groove at the interface between tubulin dimers on the MT lattice. The N-terminal tail domain of Ndc80 and the linker domain of EB1 proteins may

Bim1p phosphorylation affects spindle dynamics and is critical for proper midzone disassembly in vivo
As Bim1p phosphorylation occurred preferentially in anaphase, we compared the spindle elongation kinetics of WT and mutant bim1 cells using time-lapse live cell microscopy in strains expressing GFP-tubulin. Spindle elongation in WT cells occurred with typical biphasic kinetics consisting of an initial fast and a subsequent slow phase (Fig. 6 A). In a bim1 deletion mutant, elongation started from a shortened metaphase spindle, the rate of elongation in anaphase was reduced, and the final spindle length was decreased. The bim1 mutant mimicking Ipl1 phosphorylation (Bim1 6D ) displayed spindle elongation kinetics very similar to WT cells. In contrast, the mutant preventing Ipl1 phosphorylation (Bim1 6A ) showed a faster rate of spindle elongation in both phases of anaphase B and reached an increased maximum spindle length. These results suggest that Bim1 phosphorylation is required for spindle elongation to occur with normal kinetics. As the spindles in the phosphorylation-deficient bim1 mutant grew faster and overextended, we also analyzed the mode of spindle disassembly by following the dynamic behavior of GFP-tagged Bim1 WT or Bim1 phosphorylation mutants in strains expressing mCherry-tubulin. Bim1 WT was prominently localized to the spindle midzone in anaphase cells, and during spindle disassembly, the length of the Bim1 zone gradually decreased until only a small amount of Bim1p remained on the ends of depolymerizing spindle MTs (Fig. 6 B and Video 6). The Bim1 phosphorylation-deficient mutant displayed a different behavior: upon spindle disassembly, the length of the Bim1 midzone decreased slightly, but in contrast to WT cells, a significant amount of Bim1 6A remained on the spindle. Instead of disassembling by depolymerization, the spindle then broke, and Bim1p remained on the ends of the MTs (Fig. 6 B, right; and Video 7). In extreme cases, instead of disassembling, the spindle even started to polymerize again and was bent before finally separating (Video 8). The Bim1 6D mutant displayed only a weak signal on the spindle MTs, and the spindle disassembled efficiently (Video 9). Collectively, these results demonstrate that Bim1 phosphorylation by Ipl1 is required for efficient disassembly of the spindle midzone in vivo.

Discussion
The mitotic kinase Aurora B has an important role in regulating MT plus ends in the process of kinetochore biorientation and at the spindle midzone, but only few relevant substrates have been identified. In this study, we show that Bim1p, a member of the evolutionary conserved family of EB1 proteins, is a physiological substrate of the yeast Aurora kinase Ipl1p. In addition to characterizing Bim1's role in spindle dynamics, our study provides insights into the molecular mechanism and regulation of autonomous plus end tracking by EB1 proteins. flow cell. In the kymograph representation, the Bim1p signal disappears rapidly from the stable MTs; in contrast, it persists in a buffer control (Video 4). (E) In vitro tracking of Bim1p in the presence of the Ipl1-Sli15 complex. Bim1p is removed from growing MT ends when the Ipl1-Sli15 complex and ATP are present (Video 5). Bar, 5 µm.
to combine them with adjacent flexible regions that have an overall positive charge. Modulating the electrostatic properties of the linker domain may be a very effective way to control the affinity for MTs. Mechanistically, there are different possibilities contribute to binding by interacting with the negatively charged C-terminal tails of -tubulin (E-hook). CH domains are used by a variety of molecules with very different binding partners. It seems that a general theme for their usage in MT binding is

Establishing molecular requirements for plus end tracking
The reconstitution of MT plus end tracking in vitro using a purified system in combination with the engineering of fluorescently labeled Bim1p variants has allowed us to address the basic molecular requirements for autonomous tip tracking. We demonstrate that Bim1p, like Mal3 (Bieling et al., 2007), autonomously tracks growing MT ends in a purified system. Given the high degree of evolutionary conservation and a recent study demonstrating end tracking of Xenopus laevis EB1 (Bieling et al., 2008), we expect autonomous tip tracking to be a general, defining feature of all EB1 proteins. Furthermore, we show that MT-binding CH domains and even dimerized CH domains alone are not sufficient to allow effective plus end tracking of Bim1p in vitro. Instead, our results indicate that the flexible linker region has an essential role in providing full MT-binding and tip-tracking activity. The failure of a monomeric Bim1p construct to display tip tracking is not just caused by its overall lower binding affinity for MTs because even raising its concentration in the assay does not lead to a specific accumulation at MT ends. Instead, it seems that the dimeric arrangement of CH and linker domains is essential for the effective recognition of a specific structure at assembling MT ends (Bieling et al., 2007). This result agrees with in vivo observations demonstrating that artificial dimerization allows plus end tracking of various +TIPs (Slep and Vale, 2007). However, it is in contrast to a recent study demonstrating that an isolated CH domain of the human EB3 protein shows specific accumulation on growing plus ends in vitro (Komarova et al., 2009). Different properties of Bim1p versus EB3 may explain the contrasting experimental outcomes. In the future, the minimal requirements for plus end tracking will have to be investigated in more detail, and it will be interesting to see whether MT-binding proteins that have different arrangements of CH domains can display autonomous tip tracking. For example, in the Ndc80/Nuf2 head of the Ndc80 complex, the two CH domains reside very close together (Ciferri et al., 2008) and provide one common rather than two spatially separated MT-binding interfaces.

The role of Bim1 phosphorylation in vivo
What is the physiological role of Bim1 phosphorylation by Ipl1p? As judged from a mobility shift in SDS-PAGE, a fraction of Bim1p becomes phosphorylated in a narrow time window after securin degradation in mid to late anaphase. At this time, Bim1p localizes to the midzone of the anaphase spindle and is critical for its structural integrity by stabilizing the zone of overlapping MT plus ends (Gardner et al., 2008). Our results suggest that phosphorylation by Ipl1 regulates the interaction of Bim1p with the anaphase spindle. The Ipl1-Sli15 complex colocalizes with Bim1p to the spindle midzone (Khmelinskii et al., 2007), and Ipl1 kinase activity increases before spindle disassembly (Buvelot et al., 2003). Although a stable midzone is essential for proper spindle elongation, the zone of overlapping MT plus ends needs to be destabilized to allow efficient spindle disassembly in telophase. In a WT cell, the majority of Bim1 is unloaded from the spindle just before disassembly, but in the Bim1 phosphorylation mutant, this unloading process is inefficient, the midzone is hyperstable, and the spindle disassembles by which this can be achieved. First, multisite phosphorylation may down-regulate the MT-binding activity that is intrinsic to the unstructured region by changing its bulk electrostatic properties (Serber and Ferrell, 2007). Second, it may change the conformation of the flexible linker region with the consequence that the CH domains are not in the optimal position for cooperative MT binding anymore. Third, cluster phosphorylation may create a negatively charged domain that could compete with tubulin for binding to the CH domains (Fig. 7 A). The observed conformational compaction of the Bim1 molecule upon multisite phosphorylation of the linker domain supports the latter two possibilities. In vitro binding assays 5 µg of purified GST fusion Bim1 proteins was added to 30 µl glutathione-Sepharose and 10 µg of the Ipl1-Sli15 complex (precleared with the same amount of beads) in a final volume of 400 µl of binding buffer (25 mM Hepes, pH 7.5, 100 mM KCl, 3 mM MgCl 2 , 1 mM EDTA, 10% glycerol, 0.1% NP-40, and 1 mM DTT). After incubation for 1 h at 8°C, beads were washed three times with 0.5 ml of binding buffer and analyzed by Western blotting, and kinase complex was detected with anti-5× His antibodies (QIAGEN).

Analytical SEC
The indicated Bim1 variants were diluted into gel filtration buffer (25 mM NaH 2 PO 4 /Na 2 HPO 4 , pH 6.8, 150 mM NaCl, and 1 mM EDTA) on ice for 10 min before loading onto a Superdex 200 PC 3.2/30 (GE Healthcare) or Superose 6 PC 3.2/30 column (GE Healthcare) equilibrated in gel filtration buffer. Fractions of 50 µl were collected and separated by SDS-PAGE. Proteins were stained with Coomassie Brilliant blue R250.

MT-binding assays (conventional/fluorescence based)
Binding assays were performed essentially as previously described (Cheeseman et al., 2001). Precleared Bim1 WT , Bim1 WT phosphorylated preparatively by Ipl1, and Bim1 6D (at 1 µM) were incubated with increasing concentrations of taxol-stabilized MTs (0-10 µM) in 25 mM Hepes, pH 7.5, 150 mM KCl, 4 mM MgCl 2 , 1 mM EGTA, and 10% glycerol. Bim1 bound to MTs was separated from unbound fraction by ultracentrifugation. The amount of Bim1p in supernatant and pellet was quantified by densitometry of Coomassie-stained SDS-PAGE gels using ImageJ software (National Institutes of Health). To determine apparent K d of Bim1 for MTs, data points from three independent experiments were fitted into the equation Y = 0.5 × {(K d + B max + X)  [(K d + B max + X) 2  (4B max X)] 1/2 } using Prism 4.0 (Graph-Pad Software, Inc.), where Y is the concentration of Bim1 partitioning to the pellet with the MTs, B max is the maximal fractional Bim1-MTs complex, K d is the dissociation constant, and X is the concentration of polymeric tubulin.
In the fluorescent-based MT-binding assay, 1 µM of purified Bim1 constructs was subjected to MT binding as described in the previous paragraph and centrifuged. The amount of Bim1 bound to MTs was measured fluorometrically using a plate reader (Synergy 2; BioTek), and averaged data from three individual experiments were similarly plotted as binding affinity curves.
Light-scattering assay 8 mg/ml tubulin and 1 µM of Bim1 variants were incubated on ice in BRB80 buffer containing 1 mM GTP (in a total volume of 60 µl) and transferred into 3-mm quartz cuvettes. Right-angle light scattering of the solution was recorded every 10 s for a period of 30 min at 350 nm in a spectrofluorometer (FluoroMax4; Horiba Scientific) equilibrated to 30°C.
In vitro reconstitution of plus end tracking using TIRF microscopy The preparation of perfusion chambers for microscopy was previously described (Westermann et al., 2006). Assay chambers were constructed using acid-washed cover glasses and silanized glass slides. Assembled chambers were first washed with miliQ water. A solution of 5 mg/ml biotinylated BSA (Vector Laboratories) was flowed in for 1 h and exchanged afterward with blocking solution containing 80 µg/ml casein, 0.1% pluronic F-127 (Sigma-Aldrich), and 1% polyethylene-block poly(ethylene glycol) (Sigma-Aldrich) in BRB80 buffer for 30 min. The chamber was then incubated with 0.3 mg/ml avidin DN (Vector Laboratories) in blocking buffer for at least 30 min. Reaction chambers were equilibrated with assay buffer (BRB80 supplemented with 150 mM KCl, 0.33 mg/ml casein, 0.5% -mercaptoethanol, 4.5 µg/ml glucose, 200 µg/ml glucose oxidase, and 35 µg/ml catalase) and incubated with rhodamine-labeled, GMPCPP-stabilized short MT seeds for 1 min. Dynamic MT growth was initiated by flowing 15 µM of tubulin solution (containing 1 µM rhodamine-labeled tubulin) in assay buffer and visualized at 30°C using the TIRF3 microscopy system (Carl Zeiss, Inc.) operated with Axiovision software (Carl Zeiss, Inc.) and a 100×  Plan-Apochromat 1.46 NA objective. Time-lapse recordings were acquired every 5 s using an electronmultiplying charge-coupled device camera (C9100-02; Hamamatsu Photonics). For the observation of Bim1 plus end tracking, 70 nM EGFP-tagged Bim1 protein or 150 nM Bim1-Alexa Fluor 488 was included in the tubulin mix. In phosphoregulation assays, recombinant Ipl1-Sli15 complex at 0.1 mg/ml and 0.1 mM ATP were additionally included.

Live cell imaging
To quantify spindle elongation kinetics, cells were arrested with  factor, released into synthetic medium, and imaged on concanavalin A-coated culture dishes (Matek) at 30°C. z stacks (eight stacks 0.4 µm apart) were acquired at 1-min intervals on a microscope (Axiovert 200M; Carl Zeiss, by breakage rather than plus end depolymerization (Fig. 7 B). A constitutively high level of Bim1p on the spindle would also explain why the spindle overextends in the phosphorylationdeficient mutant. With an extended overlap zone, motors can act more efficiently in the fast phase of elongation, and sustained plus end polymerization will allow further extension in the slow phase. It is important to note that the bim1 phosphorylationdeficient mutant does not precisely phenocopy the ipl1-2 mutant, as this allele is not reported to have an effect on anaphase B kinetics (Buvelot et al., 2003). This may be explained by the presence of additional Ipl1 substrates relevant for spindle elongation such as the bundling protein Ase1p (Kotwaliwale et al., 2007). We also do not exclude additional functions for Bim1 phosphorylation and for the Bim1-Ipl1 interaction, particularly in the process of kinetochore biorientation. Additional mutants that specifically affect the interaction between Bim1p and Ipl1 will be required to clarify whether this association is critical for the regulation of MT plus ends in other cellular situations.

Materials and methods
Yeast strain construction, purification of Bim1-S-Tag-tobacco etch virus (TEV)-ZZ from yeast extracts, and mass spectrometry analysis Yeast strains (Table S1) were constructed using standard procedures. C-terminal S-Tag-TEV-ZZ (tandem affinity purification) tags, deletions, and conditional alleles of Bim1 were constructed by PCR as described previously (Cheeseman et al., 2002). Phosphorylation site mutants were generated using QuikChange Multi Site-Directed Mutagenesis (Agilent Technologies), cloned into modified pRS303-3× GFP, and integrated into a bim1 strain. All growth experiments were conducted in YPD (YP + 2% dextrose), and tandem affinity purification tag purification was performed as described previously (Cheeseman et al., 2002). Mass spectrometry analysis was performed after in-solution digest of purified Bim1p from two independent preparations.