text:
Simultaneous evolutionary expansion and constraint of genomic heterogeneity in multifocal lung cancer

Recent genomic analyses have revealed substantial tumor heterogeneity across various cancers. However, it remains unclear whether and how genomic heterogeneity is constrained during tumor evolution. Here, we sequence a unique cohort of multiple synchronous lung cancers (MSLCs) to determine the relative diversity and uniformity of genetic drivers upon identical germline and environmental background. We find that each multicentric primary tumor harbors distinct oncogenic alterations, including novel mutations that are experimentally demonstrated to be functional and therapeutically targetable. However, functional studies show a strikingly constrained tumorigenic pathway underlying heterogeneous genetic variants. These results suggest that although the mutation-specific routes that cells take during oncogenesis are stochastic, genetic trajectories may be constrained by selection for functional convergence on key signaling pathways. Our findings highlight the robust evolutionary pressures that simultaneously shape the expansion and constraint of genomic diversity, a principle that holds important implications for understanding tumor evolution and optimizing therapeutic strategies.

The authors perform detailed whole exome (and some whole genome) DNA sequence analysis of lung adenocarcinomas arising in multiple synchronous lung cancers (MSLCs) in 4 female never smokers. They then compare the mutations representing coding changes in tumors from the same individual. They show by analyses of silent mutations that the lung cancers are indeed independent of one another and not metastatic lesions from the same tumor. Their key conclusions are that despite having different driver mutations the tumors from each patient appear to "converge" on the signaling pathways affected. They provide functional tests in preclinical models for several of the driver mutations found to show oncogenic activity including the ability to rescue CRISPR as9 mediated deletion of several driver mutations in lung cancer cell lines. Several xenograft studies treated with various targeted therapies targeted at the signally pathways involved were found to respond. Finally, they present data on genomic analyses of lung and breast cancers found in the same patient. While these had entirely different drive mutations they also showed similar signaling pathways affected.

Comments to the Authors:
This is a technically well done genomic analysis of an "experiment of nature" that shows the "expansion" (of genetic changes) and "convergence" (of signaling changes) in MSLCs. This paper is reviewed in the context of a very similar publication in Nature Communication in 2016 (their reference 28) which studied 17 lung adenocarcinomas in 6 patients (4 never smokers and 2 former smokers). The ethnicity of patients studied in both the current paper and the reference 28 paper are likely to both by East Asian. Thus, the genomic findings in the two studies are on almost identical tumors and tumor settings and the main results appear to be very similar. The current study provides some functional data which the other study does not. There are several things that would have greatly improved the current report. 1. The main paper needs a simple table (that currently is given in the supplemental section) on the age, gender, ethnicity, smoking status and presence of well known driver mutations in the tumors from the 4 patients. If other data needs to be moved to the supplemental section to accommodate this that is fine. The reader should not have to go to the supplemental sections to find these key pieces of information. 2. If they had information on MSLCs in at least one smoker their (which is the most common type of lung cancer) their case would have been strengthened. 3. Given their arguments of convergence of pathways, I expected to see multiple immunohistochemical (IHC) sains testing a variety of signaling pathways known to be altered in lung adenocarcinomas (other than ERK) to functionally prove in these patients that signaling pathways actually expressed were indeed constrained. Similarly, IHC stains of adenocarcinoma lineage oncogenes and differentiation markers (like TTF1 and napsin) to see how these compared between the MSLCs would have been very helpful. 4. We know the tumor microenvironment plays a key role in lung cancer pathogenesis including potentially immune responses. So one questions is whether the neoantigen load was similar in the different MSLCs and was there any evidence of immune selection similarities or differences in tumors from the same patient? 5. A part of the same analyses, IHC stains for key components of the tumor microenvironment would have been very helpful. Whatever the answers are (similar or different for the MSLCs) they would be important to know. In fact, such information would have huge impact on planning for precision immune treatment regimens as much or perhaps more than for targeted therapy. 6. Do "branched" or multi-region analyses within a tumor show the same kind of convergence? In the paper's current format, I am not sure what the authors' conclusions are from the analyses presented. 7. While other molecular studies are perhaps the subject of future research, I would have bee interested to know the status of epigenetic changes and RNA expression profiles in the different tumors from the same patient. Such information could have also provide answers to the questions posed above such as information on signaling pathways and the tumor micro-environment. The epigenetic studies would give us important information on whether they who the same expansion and convergence that the mutational studies show.
8. Whey was the 5th patient reported separately? This seems unclear.
Reviewer #2: Remarks to the Author: The authors present a multi-region sequencing analysis of a unique cohort of multifocal NSCLCs. Both the sample set and the functional validation are very interesting. However there are a few major issues that would need to be addressed: 1) The phenotypic/functional convergence between lesions from the same patient is not surprising. Mutations in EGFR (alone) account for 10-35% of NSCLC and the whole pathway could amount to up to 50%. Similarly, KRAS+BRAF+etc can be as high as 35%. Without a large patient cohort, it is not surprising that two or three co-occurring tumours are driven by the same pathway. This issue would be compounded if the patients in the study were selected from a larger study cohort. Hence the conclusions in the manuscript need to be tempered.
2) The 'schematic model' in figure 5e makes very little sense and does not add anything to the discussion.
3) Fig. 2c: Despite the importance of protein expression changes when KRAS, BRAF, MEK1 were modified in HEK293T and LXF-289 cell lines, the same has not been shown in BEAS-2B cell line. This is surprising since BEAS-2B cell line was chosen for two important assays ( Fig. 2d and 2e) and to make a strong argument for their case. It will be important to demonstrate the same panel of protein expression changes in modified BEAS-2B cells before moving to in vivo. Fig. 2c: pERK expression seems to be differently regulated in HEK293T and LXF-289 cell lines when D594G modification was introduced. It is surprising to see that the authors performed an experiment using human kidney cell line and made a comparison with lung adenocarcinoma cell line. The latter shows increased kinase activity in lung adenocarcinoma cell line with BRAF D594G modification as assessed by western blot analysis. This difference needs to be discussed in more detail and the authors need to take the increase of pERK expression in BRAF D594G modified LXF-289 cells into consideration. Fig. 2d: It would make a stronger argument if they have performed the colony formation assay in at least KRAS modified BEAS-2B cell line.

Reviewer #1 Expert in lung cancer genomics: Comments to the Author
The authors perform detailed whole exome (and some whole genome) DNA sequence analysis of lung adenocarcinomas arising in multiple synchronous lung cancers (MSLCs) in 4 female never smokers. They then compare the mutations representing coding changes in tumors from the same individual. They show by analyses of silent mutations that the lung cancers are indeed independent of one another and not metastatic lesions from the same tumor. Their key conclusions are that despite having different driver mutations the tumors from each patient appear to "converge" on the signaling pathways affected. They provide functional tests in preclinical models for several of the driver mutations found to show oncogenic activity including the ability to rescue CRISPR as9 mediated deletion of several driver mutations in lung cancer cell lines. Several xenograft studies treated with various targeted therapies targeted at the signally pathways involved were found to respond. Finally, they present data on genomic analyses of lung and breast cancers found in the same patient. While these had entirely different drive mutations they also showed similar signaling pathways affected.
Comments to the Authors: This is a technically well done genomic analysis of an "experiment of nature" that shows the "expansion" (of genetic changes) and "convergence" (of signaling changes) in MSLCs. This paper is reviewed in the context of a very similar publication in Nature Communication in 2016 (their reference 28) which studied 17 lung adenocarcinomas in 6 patients (4 never smokers and 2 former smokers). The ethnicity of patients studied in both the current paper and the reference 28 paper are likely to both by East Asian. Thus, the genomic findings in the two studies are on almost identical tumors and tumor settings and the main results appear to be very similar. The current study provides some functional data which the other study does not. There are several things that would have greatly improved the current report.
1. The main paper needs a simple table (that currently is given in the supplemental section) on the age, gender, ethnicity, smoking status and presence of well known driver mutations in the tumors from the 4 patients. If other data needs to be moved to the supplemental section to accommodate this that is fine. The reader should not have to go to the supplemental sections to find these key pieces of information.
We thank the reviewer's suggestions, and have added a table (originally Supplementary  Table 1) to provide the clinical information of the 4 patients with MSLC. The driver mutations for each tumor are shown in Fig. 3a. 2. If they had information on MSLCs in at least one smoker their (which is the most common type of lung cancer) their case would have been strengthened.
We agree with the reviewer that it would be nice to have included more MSLCs in our cohort, especially those with smoking history. Unfortunately, however, we found that most MSLC patients eligible for surgery were non-smokers with early-stage disease. In addition, it was a priority to preserve intact tumor tissues for accurate pathological diagnosis, which made sample collection of MSLCs particularly difficult. Consequently, we were not yet able to obtain appropriate MSLC specimens from smokers. An expanded, multicenter clinical investigation of MSLCs is underway and we hope to address this question in the future.
3. Given their arguments of convergence of pathways, I expected to see multiple immunohistochemical (IHC) sains testing a variety of signaling pathways known to be altered in lung adenocarcinomas (other than ERK) to functionally prove in these patients that signaling pathways actually expressed were indeed constrained. Similarly, IHC stains of adenocarcinoma lineage oncogenes and differentiation markers (like TTF1 and napsin) to see how these compared between the MSLCs would have been very helpful.
Following the reviewer's advice, we have performed immunohistochemical staining of phospho-AKT (T308), Napsin A and TTF-1. These data have been included in the revised manuscript ( Supplementary Fig. 13a). 4. We know the tumor microenvironment plays a key role in lung cancer pathogenesis including potentially immune responses. So one questions is whether the neoantigen load was similar in the different MSLCs and was there any evidence of immune selection similarities or differences in tumors from the same patient?
We thank the reviewer for the great suggestions. Neoantigens were predicted for each tumor of MSLCs using NetMHCpan algorithm, which revealed distinct repertoires and unequal load of neoantigens associated with different lesions (Supplementary Fig. 9b; Supplementary Table 5). These results implied functionally heterogeneous tumorassociated lymphocytes and potentially differential clinical responses to immune treatment regimens among MSLC loci. We have included these data in the revised manuscript. 5. A part of the same analyses, IHC stains for key components of the tumor microenvironment would have been very helpful. Whatever the answers are (similar or different for the MSLCs) they would be important to know. In fact, such information would have huge impact on planning for precision immune treatment regimens as much or perhaps more than for targeted therapy.
To address the heterogeneity of tumor microenvironment, CD3 IHC was conducted to probe the T cell population. Interestingly, consistent with a recent report, all tumor sections of MSLCs exhibited positive staining of CD3 ( Supplementary Fig. 9a), suggesting that T cells might already infiltrate into tumors even in early-stage lung cancer. The functional diversity of these immune cells warrants further investigations in future studies. 6. Do "branched" or multi-region analyses within a tumor show the same kind of convergence? In the paper's current format, I am not sure what the authors' conclusions are from the analyses presented.
As shown in Fig. 2f and Fig. 2g, the putative driver alterations for each region were mostly present in all sequenced loci, and thus represent truncal/early somatic mutations. Since there were very few driver events on the branches, we did not observe the same kind of convergent evolution as in MSLCs. Previous reports have indicated that most known driver mutations occur early in lung tumor evolution. On the other hand, studies in clear cell renal cell carcinoma (Nat Genet. 2014 Mar;46(3):225-33.) and glioma (Nat Genet. 2015 May;47(5):458-68.) did indeed find typical examples of parallel mutations with multi-region analyses. It should be noted that the subclones in those studies originated from the same tumor progenitors, and therefore were fundamentally different from multiple synchronous thoracic lesions which were completely independent as described in our manuscript. 7. While other molecular studies are perhaps the subject of future research, I would have bee interested to know the status of epigenetic changes and RNA expression profiles in the different tumors from the same patient. Such information could have also provide answers to the questions posed above such as information on signaling pathways and the tumor micro-environment. The epigenetic studies would give us important information on whether they who the same expansion and convergence that the mutational studies show.
We agree with reviewer that the status of epigenetic changes and RNA expression profiles in different tumors from the same patient would be quite informative to illuminate the evolutionary principles of MSLC. However, with very limited tumor materials available, we were only able to perform genomic DNA sequencing as the first step to delineate the molecular underpinnings of MSLC development. Epigenetic or transcriptomic analyses require high-quality clinical samples and yet-to-be-improved quantitative approach to deal with small cohort size, which, at the moment, is extremely challenging and beyond the scope of our current project. We appreciate the reviewer for the insightful comments, but feel that such studies could be subjects of future reports. 8. Whey was the 5th patient reported separately? This seems unclear.
We used the first 4 MSLC patients (RJLC1-4) as the discovery cohort, and the last 3 (RJLC5-7) patients as the validation cohort. We collected fresh tumor samples upon surgery from each lesion of RJLC1-4, and these tissues were subjected to whole-exome sequencing for the initial genomic characterization of MSLCs. RJLC5-6 harbored a tumor in breast and a tumor in lung, and thus represented a different, but related, scenario. RJLC7 was a special case, who underwent surgical resection of two MSLCs in her right lobes and received gefitinib treatment to control the remaining tumor nodes in the left upper lobe. Therefore, these analyses provided several complementary lines of evidence to support the evolutionary principle of MSLC development that we proposed.

Reviewer #2 Expert in tumour heterogeneity: Comments to the Author
The authors present a multi-region sequencing analysis of a unique cohort of multifocal NSCLCs. Both the sample set and the functional validation are very interesting. However there are a few major issues that would need to be addressed: 1) The phenotypic/functional convergence between lesions from the same patient is not surprising. Mutations in EGFR (alone) account for 10-35% of NSCLC and the whole pathway could amount to up to 50%. Similarly, KRAS+BRAF+etc can be as high as 35%. Without a large patient cohort, it is not surprising that two or three co-occurring tumours are driven by the same pathway. This issue would be compounded if the patients in the study were selected from a larger study cohort. Hence the conclusions in the manuscript need to be tempered.
The reviewer raised a critical point. It is notable that except EGFR and KRAS, other driver mutations identified in our study were diverse and relatively rare in NSCLC. These included BRAF D594G , c-MET ΔE14 , ARAF S214C or MEK1 E102-I103 del and HER2 YMVA , which led us to believe that evolutionary constraints caused this striking convergence phenomenon. However, as the reviewer suggested, we cannot completely rule out the possibility that the functional recurrent patterns in some cases may be due to coincidence, and we have toned down our conclusions in the revised manuscript.
2) The 'schematic model' in figure 5e makes very little sense and does not add anything to the discussion.
We apologize for the confusion caused by insufficient explanation. To summarize the findings of our detailed genomic analyses in MSLCs, we proposed this schematic model in Fig. 4e for open discussion, in which we believed that simultaneous evolutionary expansion and constraint of genomic heterogeneity collaboratively shaped tumorigenic process and clonal architecture. While many studies have focused on the substantial tumor heterogeneity as a result of stochastic mutations, very little attention has been given to the evolutionary constraints during tumorigenesis. Therefore, it might be important, in our opinion, to highlight the dueling evolutionary forces that control the formation of fittest tumor clones, in order to thoroughly understand tumor evolution and rationally optimize therapeutic strategies.
3) Fig. 2c: Despite the importance of protein expression changes when KRAS, BRAF, MEK1 were modified in HEK293T and LXF-289 cell lines, the same has not been shown in BEAS-2B cell line. This is surprising since BEAS-2B cell line was chosen for two important assays (Fig. 2d and 2e) and to make a strong argument for their case. It will be important to demonstrate the same panel of protein expression changes in modified BEAS-2B cells before moving to in vivo.
We thank the reviewer for the great point. The impact of wild-type or mutant KRAS, BRAF, MEK1 expression on downstream MAPK pathway in BEAS-2B cells has been determined and the results were shown in revised Fig. 2c. All these putative driver oncogenic mutations led to ERK hyperactivation. Fig. 2c: pERK expression seems to be differently regulated in HEK293T and LXF-289 cell lines when D594G modification was introduced. It is surprising to see that the authors performed an experiment using human kidney cell line and made a comparison with lung adenocarcinoma cell line. The latter shows increased kinase activity in lung adenocarcinoma cell line with BRAF D594G modification as assessed by western blot analysis. This difference needs to be discussed in more detail and the authors need to take the increase of pERK expression in BRAF D594G modified LXF-289 cells into consideration.

4)
D594 resides in the highly conserved DFG motif of BRAF kinase domain. As a result, mutations of this residue, including D594G or D594N described in our manuscript, are catalytically inactive, which is consistent with reduced phospho-ERK in HEK293T cells associated with BRAF D594G as compared to BRAF WT . Counterintuitively, D594 mutants have been frequently discovered in various human cancers (http://www.cbioportal.org/). Heidorn et al proposed that these kinase-dead mutants was proficient in binding with CRAF and consequently driving MEK/ERK activation in the presence of oncogenic RAS (Cell. 2010 Jan 22;140(2):209-21.). We speculate that this scaffold mechanism of BRAF D594G to promote RAS-CRAF signaling underlies the increased phospho-ERK and transforming phenotype in BEAS-2B or LXF-289 cells. Therefore, the effects of BRAF D594G on MAPK pathway activity are context-dependent, and we have discussed this point in more detail in the revised manuscript. Fig. 2d: It would make a stronger argument if they have performed the colony formation assay in at least KRAS modified BEAS-2B cell line.

5)
We have attempted to perform the colony formation assay in BEAS-2B and LXF-289 cells overexpressing KRAS WT or KRAS G12D . Consistent with previous reports, we found that KRAS G12D expression triggered cellular senescence in vitro, which confounded the evaluation of its oncogenic potential using this assay (Please see the images shown below). Considering that KRAS G12D is a well-characterized oncogene, and that our focus was two novel candidate driver alterations (BRAF D594G and MEK1 E102-I103 del ), we did not discuss these results in our manuscript. 6) Fig. 2e: This experiment is lacking a very important point where the comparison between WT KRAS, BRAF, MEK1 and modified versions has not been performed while this has been clearly demonstrated in Fig.2c and 2d. This is critical as the main message of this figure should be the difference between WT KRAS, BRAF, MEK1 and modified versions rather than with empty vector.
We have performed the xenograftation experiment for BEAS-2B cells expressing wildtype KRAS, BRAF, MEK1, and found that none of these cells were able to form palpable tumors within the same time frame (Please see the images shown below). The manuscript has been revised to include these controls (Fig. 2e).


extfieldsofstudy: Biology Medicine

text:
The Contribution of Neuromuscular Stimulation in Elucidating Muscle Plasticity Revisited

Studies carried out during the past 45 years on the effects of chronic low-frequency stimulation on skeletal muscle have revealed a multiplicity of adaptive changes of muscle fibres in response to increased activity. As reflected by induced changes in the metabolic properties, protein profiles of the contractile machinery and elements of the Ca2+-regulatory system, all essential components of the muscle fibre undergo pronounced changes in their properties that ultimately lead to their reversible transformation from fast-to-slow phenotype. The chronic low-frequency stimulation experiment thus allows exploring many aspects of the plasticity of mammalian skeletal muscle. Moreover it offers the possibility of elucidating molecular mechanisms that remodel phenotypic properties of a differentiated post-mitotic cell during adaptation to altered functional demands. The understanding of the adaptive potential of muscle can be taken advantage of for repairing muscle damage in various muscle diseases. In addition it can be used to prevent muscle wasting during inactivity and aging. Indeed, pioneering studies are still the sound grounds for the many current applications of Functional Electrical Stimulation and for the related research activities that are still proposed and funded.

The present paper is not a comprehensive review but it was written to illustrate findings that show the dynamic state of skeletal muscle fibers. Early studies on rabbit soleus muscle had shown that the maintenance of phenotypic properties depended on activity. 1 Further development of this observation lead to experiments that used externally applied electrical stimulation. 1,2 The application of this technique provided an opportunity to study the phenotypic adaptability of the terminally differentiated muscle fibre. The purpose of this article is not to provide a review of all the observations made since but rather to show the evolution of ideas that have contributed to the notion of muscle plasticity. The term plasticity of muscle has been coined during the preparation of an international symposium held at the University of Konstanz in September 1979. 3 The topics that were treated included the heterogeneity of muscle as a tissue, the programme of its ontogeny and differentiation, and the regulation of its properties by innervation, use, hormones and modulation of neural input. The state of the art on these subjects was such that the time was ripe to bring together scientists from various disciplines to discuss these issues.
It has been known for a long time that mammalian muscle fibres are not all the same but differ in their various properties. [4][5][6] The problem of understanding the significance of this diversity for the organization of motor control had been highlighted in the last century by studies of Henneman and his colleagues who discovered that motoneurones supplying particular types of muscle fibres are specialized. 7 Those motoneurones that supply slow and fatigue resistant muscle fibres have a lower threshold and are more active, whereas motoneurones to muscle fibres that contract and relax fast have a high threshold and are therefore less active. The relationship between the motoneurone and muscle fibres it supplies was most clearly illustrated by Eccles and co-workers in the early 60th by their cross reinnervation experiments. 8 The mechanism as to how motoneurones control muscle properties was not clear at that time but has been attributed to a special trophic influence of the motor nerve on muscle. An alternative proposal that could explain these observations, was that re-innervating the muscle with an alien nerve, had altered its activity pattern. This interpretation was supported by tenotomy experiments on rabbit soleus muscle which changed the activity pattern of the muscle without altering its innervation. Returning the continuous activity of the soleus nerve to the silenced tenotomised muscle by electrical stimulation restored the slow time course of contraction. 1 It was concluded from these experiments that the continuous impulse activity normally present in postural soleus muscle, is responsible for the slow time course of contraction and relaxation of this muscle. Due to the initiative of Ernest Gutmann and Sidney Hilton, Gerta and Dirk met at this stage, and decided to join forces to elucidate this programme. Their encounter and collaboration turned out to be chance and necessity. The data reported here is mainly derived from that collaboration and should not be considered a comprehensive review as most of this has been covered by several reviews. [9][10][11] Earlier studies on energy metabolism in functionally distinct muscles had led to the definition of specific and constant proportions in the enzyme activity patterns. These findings supported the notion that energy supply is adjusted to specific functional demands of different muscles. 12 The analysis of enzyme activity patterns, therefore, represented an appropriate tool to investigate metabolic specialization of muscles and other tissues. 13 The application of this approach to cross re-innervated muscles in rabbit showed pronounced changes in the enzyme patterns matching the induced functional changes. 14 Similar metabolic changes were described in cross re-innervated muscles of cat and rat. [15][16][17] Nevertheless, it was clear that these experiments did not provide an answer that distinguished between the hypotheses of trophic factors or activity regulating muscle properties. The development of new implantable materials then allowed examining the hypothesis that activity is responsible for determining muscle properties by means of the chronic stimulation experiment. Electrodes were implanted either side of the peroneal nerve in one leg of anesthetized rabbits. The electrodes were then connected to a stimulating device and impulses of supramaximal intensity at 10 Hz were delivered to the nerve. This frequency pattern resembled that of motoneurones innervating the soleus muscle. This did not cause discomfort to the animal. Contractile properties of the stimulated tibialis anterior (TA) and extensor digitorum longus (EDL) muscles were examined at various time intervals using conventional physiological methods. Within two to four weeks, the stimulated muscle became more slowly contracting. 1,2 At this stage Gerta and Dirk started their collaboration and together they examined enzyme profiles of stimulated and control muscles. In these studies, a profound re-arrangement of enzyme activity and isozyme patterns confirmed that prolonged slow frequency (10 Hz) activity imposed on fast muscles that were normally getting their energy from breakdown of glycolytic pathways, induced their transformation towards a slow-oxidative muscle. At that time, the interpretation of our results was not readily accepted by the scientific community that favoured the idea of trophic factors released from the nerve being responsible for determining muscle properties .8 Even at that time this view was challenged by A.F. Huxley who suggested that the slow frequency activity of slow motoneurones induces a vibratory stress to fast muscles that elicits the change from fast to slow phenotype (l.c. Buller et al. 1960). 8 The dramatic transformation of adult differentiated fast glycolytic muscle fibres into slow oxidative ones by altered activity inspired us to use this experimental model to study not only the potential of this tissue to adjust to activity but also to unravel the mechanisms by which such changes are induced.

Chronic low-frequency stimulation as an experimental model
In addition to establishing the physiological importance of activity as a decisive factor in determining muscle properties, chronic nerve stimulation of fast muscle at low frequency provided an excellent model for studying the time course of activity-induced changes. In other words, the stimulation experiment allowed us to follow changes of cellular re-organization in a differentiated post-mitotic tissue during its transition from a relatively stable to an altered state induced by new functional demands. It also enabled us to investigate both, the hierarchy and the sequence of changes at different levels of cellular organization and, establish the full range of adaptation during the transition from a fast to a slow muscle, and finally propose a mechanism that initiated this change. Another advantage of the stimulation experiment was that all motor units of the target muscle were simultaneously activated by the same pattern of activity. The unstimulated contralateral muscles served as an intra-animal control. Systemic influences such as stress, changes in hormone homeostasis, etc., would thus equally affect stimulated and contralateral control muscles. Analyses of time-dependent changes of chronic low-frequency stimulation (CLFS), demanded standardization of the experimental set-up so as to create reproducible conditions regarding patterns, intensity and duration of activity transmitted to the target muscle. This was achieved by developing a method of tele-stimulation. 18 In the following text, effects of CLFS on selected properties of muscle fibres in response to this new type of activity are described. Apart from functional tests, our studies included energy metabolism, contractile and regulatory proteins of the myofibrillar apparatus and the sarcoplasmic reticulum. These objects were chosen in the hope to elucidate both the hierarchy and sequence of changes that would help to explain the mechanisms underlying the adaptive potential of the muscle.

Metabolic transformation
The metabolic transformation of rabbit fast-twitch muscles (EDL, TA) exposed to low-frequency stimulation (CLFS) was characterized by increases of enzyme activities involved in aerobic-oxidative metabolism and simultaneous decreases in glycogenolytic and glycolytic metabolic capacities. [19][20][21][22][23] Twenty eight days after the onset of stimulation glycogenolytic and glycolytic capacities decreased to ~40% of their control values, while there was an almost 8-fold increase in mitochondrial volume density. The relative activities of soluble and membrane-bound enzymes of aerobic oxidative metabolism (the citric acid cycle, fatty acid oxidation) remained unaltered although their absolute activity levels increased by 5-8fold. 22 An even greater increase was seen in ß-hydroxybutyrate dehydrogenase, a structure-bound mitochondrial enzyme. In contrast, glycerolphosphate oxidase, another mitochondrial structure-bound enzyme representing the mitochondrial part of the glycerolphosphate shuttle, markedly decreased and paralleled the changes in glycolytic capacity. These results taken together, suggest that not only was there an increase in mitochondria but also a change of their composition. Stimulation of fast muscles also induced an early and pronounced increase in capillary density. 24 The study of the time course of this increase compared to that of succinate dehydrogenase (a marker for mitochondria) revealed that the change in capillary density preceded that in aerobic-oxidative capacity. 25 An improved oxygen supply provided by the capillaries thus might have induced the increase in mitochondria.

Contractile proteins
The altered time course of contraction after CLFS had indicated a change in the contractile machinery of the muscle. This was confirmed by qualitative histochemistry of myofibrillar ATPase activity. Stimulated muscles displayed a greater proportion of fibres histochemically classified as slow (type I). 20,21,26 Subsequent studies showed alterations in the composition of myofibrillar proteins in whole muscle and in single fibre extracts. The myosin isoform patterns, both at the levels of light (MLC) and heavy (MHC) chains were shifted towards slower isoforms. The time course of these changes in rabbit muscle suggested a sequential exchange of fast with slow isoforms in the order of MHCIId, MHCIIa, MHCI. This sequence was confirmed in single fibre studies that also showed appearance of hybrid fibre types co-expressing several MLC and MHC isoforms. 27,28 Expression patterns of MHC isoforms, both at the levels of mRNA and protein in single fibres at different stages of transformation displayed a greater variability at the mRNA than that at the protein level. 28 The coexistence of different mRNA isoforms in the same fibre was not always seen at the protein level, indicating that the switch from transcription to translation is likely to be separately controlled. Studies of the time course of MHC isoform transitions performed on rat muscle provided similar results. However in rats, the fastest fibres in EDL and TA muscles are type IIB which contain MHCIIb. 29 When exposed to CLFS, these fibres are first transformed into type IID and successively type IIA. Considering the results from both, rat and rabbit muscle, the fast-to-slow transition isoforms occurs in an orderly manner so that MHCIIb -> MHCIId -> MHCIIa -> MHCI. During these fibre transitions, isoforms initially present in the fibre were replaced by newly synthesised isoforms which resulted in the transitory appearance of hybrid fibres containing more than one MHC isoform. As documented by single fibre analysis, the complete sequence of fast-to-slow transitions was as follows: MHCIIb -> MHCIIb/IId -> MHCIId -> MHCIId/IIa -> MHCIIa -> MHCIIa/I -> MHCI. Depending on the initial phenotype of the fibre, the CLFS-induced fast-to-slow transitions differ between fibres. This has been revealed by single fibre analyses. 30 These changes are reversible as most CLFS-induced changes studied so far. The direction of the arrows in the above scheme can be reversed. Therefore, discontinuing stimulation presents the possibility for the study of slow-to-fast transitions. [31][32][33] As illustrated in studies on rat muscle, the initial change of the transformation consisted of a repression of MHCIIb. Its decay was faster at the mRNA than at the protein level. The corresponding half-lives amounted to approximately 3 days for the mRNA and 11 d for the protein which indicated a rapid re-modelling of the myofibrillar apparatus in response to increased activity. 33 Newly synthetized MHCIId and MHCIIa replacing MHCIIb, were traced in rat TA muscle by measuring protein synthesis by 35 S-methionine incorporation. Increases in synthesis of MHCIId and MHCIIa occurred as soon as two days after stimulation onset. 34 This data supported our notion that changes in MHC isoform expression are early processes in response to the altered activity

Ca 2+ -regulatory proteins
Fast-to-slow transformation included also exchanges of fast with slow isoforms of the regulatory proteins at the level of the thin filament. Due to the greater number of isoforms, especially in the case of troponin with three fast and two slow isoforms, the fast-to-slow transitions proved to be more complex. Nevertheless, the time course studies showed that the shift from fast to slow occurred in a manner coordinated with that of the MHC isoforms. 35 The major changes in the protein profile of the sarcoplasmic reticulum (SR) consisted of an exchange of the fast SERCA1 with the slow SERCA2 Ca 2+ -ATPase isoform. This exchange paralleled the fast-toslow transition at the stage from MHCIIa to MHCI. 36 The switch in SERCA isoforms was preceded by a partial (40-50%) inactivation of the ATPase activity [37][38][39] due to protein oxidation and nitration of tyrosyl groups of the enzyme. 40 Both processes may contribute to the prolonged relaxation in the stimulated muscle. In addition to these alterations of Ca 2+ -ATPase, other proteins were also affected. These included pronounced decreases (~60%) in the levels of ryanodine receptor, dihydropyridine receptor, sarcalumenin, and triadin. Calsequestrin displayed only a small decrease of ~20%. 36 Parvalbumin, a cytosolic Ca 2+ -binding protein present only in fast-twitch fibres, was decreased rapidly during CLFS. Its decay in rabbit muscle paralleled the first step (MHCIId to MHCIIa) of the fast-to-slow transition in MHC isoforms. 41 Loss of parvalbumin may, therefore, explain the early changes in relaxation speed. These early changes of proteins involved in Ca 2+ -uptake indicated alterations in intracellular Ca 2+ -dynamics. These changes might induce alterations in gene expression related to Ca 2+ -dependent signalling. Indeed, measurements of Ca 2+ transients in single fibres isolated from rat EDL muscle exposed to CLFS showed an instantaneous ~2.5-fold increase in resting Ca 2+ which was maintained during the entire period of 10 days of stimulation. 42 This value was in the range characteristic of slow-twitch fibres from soleus muscle. There was also a transitory increase of the integral Ca 2+ per pulse with a maximum (~5-fold) after 1 day. Steep decreases in the rate constant of Ca 2+ -decay could be explained by an immediate impairment of the Ca 2+ -uptake and by an additional loss of Ca 2+ -binding capacity, most likely due to the decay in parvalbumin. It has been shown that the expression of specific protein patterns in distinct muscle fibre types is under the control of Ca 2+ -dependent signalling. Sustained increases of resting Ca 2+ levels have been shown to activate transcription of slow fibre-specific proteins mediated by a calcineurin-dependent pathway. 43 An impairment of Ca 2+ -uptake in the stimulated muscle by the early partial inactivation of the SR Ca 2+ -ATPase, and the decrease in cytosolic Ca 2+ -binding capacity (parvalbumin) are likely to be responsible for the increased levels of resting Ca 2+ and the activation of molecules controlling the slow phenotype. The impairment of Ca 2+ -uptake that preceded the SERCA1 to SERCA2 transition, may, however, not only result from the above mentioned mechanisms but may also be due to a compromised energy state of the stimulated muscle fibre. In fact, measurements of the ATP phosphorylation potential [ATP/ADP free ] in whole muscle and single fibres revealed an almost instantaneous drop after stimulation onset. 28,44 The ATP phosphorylation potential remained for up to 50 days of stimulation at ~50% of that in unstimulated muscle. The drop in the phosphorylation potential was accompanied by an almost instantaneous decrease in force production which was reduced to 28% of the control immediately after the onset of stimulation and gradually recovered during the following 10 days. 44,45 The recovery of force was most likely brought about by the metabolic transformation, i.e. the improved energy supply via aerobic-oxidative pathways. Consistent with this is the finding that lowering the cellular phosphorylation potential by other means, such as by administration of a creatine analogue, has also been shown to shift myosin isoform expression towards the slow phenotype, but these changes were less complete than those brought about by CLFS. 46,47 The instantaneous effects of CLFS thus affect at least two major elementary functions of the muscle fibre, its energy state and its Ca 2+ homeostasis. These may be the initial signals effecting signalling pathways controlling the expression of phenotypic properties in various compartments of the muscle fibre. In summary, these and other results of the multiple effects of CLFS demonstrate the value of this experimental set-up for studying muscle plasticity and its regulatory mechanisms. In our opinion, this model has not been fully exploited and offers unique possibilities for studies of regulatory processes underlying muscle plasticity. Such studies need a multidisciplinary approach as shown in the present report.

Application of electrical stimulation to treatment of neuromuscular disorders
Results of experiments on mutant mice suffering from severe muscle weakness (C7BL dy/2J/dy2J), show that a particular pattern of activity imposed upon their muscles by stimulation of their nerve reduces the rate of deterioration of their muscles. These mutant mice have a defect of laminin alpha 2 (Lama 2) gene. 48 CLFS of their leg muscles lead to a reduction of the rate of loss of force and muscle bulk that is typical in these animals. 49,50 Moreover, CLFS reversed the changes in enzyme activities caused by the disease in these dystrophic mice. 51 The encouraging results obtained on animal models led to attempts to investigate the effects of CLFS in patients suffering from Duchenne muscular dystrophy. Consistent with the results on mice, the results showed that electrical stimulation reduces the rate of deterioration of ankle dorsi-flexors and quadriceps muscles, provided the stimulation was started before the muscles became excessively weak. The frequency at which the muscles were stimulated was of critical importance for only stimulation at 10 Hz or less had a beneficial effect, while frequencies of 30 Hz or more enhanced the deterioration of the muscles. 52,53 With the development of methods that attempt treatment of muscle diseases by trying to correct the genetic fault by exon skipping (Duchenne muscular dystrophy) or by interfering with the splicing mechanism of the cell by antisense oligonucleotides (SMA) it may be beneficial to induce prior to treatment a situation where the affected muscle is already undergoing changes in gene expression by CLFS-induced alterations in transcriptional and/or epigenetic regulation. Finally, other effects of electrical stimulation such as increases of capillary density and blood flow, may allow better access of any substance to target muscles. An additional change contributing to an altered responsiveness of the muscle, might relate to the increase in satellite cell activation under influence of low-frequency stimulation. 54 These few suggestions point out the possibilities of using stimulation induced changes either on their own or in combination with other therapeutic interventions for treatment of neuromuscular disorders. It is surprising that these approaches have so far not been used, particularly in view of extensive evidence that electrical stimulation of human muscle is efficient as judged by biochemical and functional criteria, 55 and has recently been so widely and successfully used for rehabilitation of various conditions. 56 These include procedures used for the maintenance of muscle function after spinal cord injury, 57 denervation, 58 and aging. 59,60 These important results also provided evidence that in human muscles electrical stimulation was able to reverse many of the degenerative changes that follow denervation. 58,61 As a consequence of these beneficial effects patients were able to use their muscles during locomotion and counteract the effects of aging and long term inactivity.

Author's contributions
Authors equally organized the draft and wrote the typescript.


extfieldsofstudy: Medicine Chemistry

text:
Mitotic spindle: kinetochore fibers hold on tight to interpolar bundles

When a cell starts to divide, it forms a spindle, a micro-machine made of microtubules, which separates the duplicated chromosomes. The attachment of microtubules to chromosomes is mediated by kinetochores, protein complexes on the chromosome. Spindle microtubules can be divided into three major classes: kinetochore microtubules, which form k-fibers ending at the kinetochore; interpolar microtubules, which extend from the opposite sides of the spindle and interact in the middle; and astral microtubules, which extend towards the cell cortex. Recent work in human cells has shown a close relationship between interpolar and kinetochore microtubules, where interpolar bundles are attached laterally to kinetochore fibers almost all along their length, acting as a bridge between sister k-fibers. Most of the interpolar bundles are attached to a pair of sister kinetochore fibers and vice versa. Thus, the spindle is made of modules consisting of a pair of sister kinetochore fibers and a bundle of interpolar microtubules that connects them. These interpolar bundles, termed bridging fibers, balance the forces acting at kinetochores and support the rounded shape of the spindle during metaphase. This review discusses the structure, function, and formation of kinetochore fibers and interpolar bundles, with an emphasis on how they interact. Their connections have an impact on the force balance in the spindle and on chromosome movement during mitosis because the forces in interpolar bundles are transmitted to kinetochore fibers and hence to kinetochores through these connections.

Introduction
Cell division is one of the most fundamental processes in the living world. At the onset of division the cell assembles a spindle (Fig. 1), a fascinating and complex micromachine made of microtubules and the accompanying proteins (McIntosh et al. 2012;Pavin and Tolic 2016;Prosser and Pelletier 2017). Spindle microtubules attach to chromosomes via kinetochores, protein complexes assembled on the centromeres of each chromosome (Musacchio and Desai 2017). The central question in the field is how the cell achieves accurate chromosome segregation through interactions between kinetochores, microtubules, and the associated proteins.
Spindles in mammalian cells contain hundreds of microtubules, which are connected in a complex fashion with the help of various microtubule-associated proteins including motor proteins and passive crosslinkers. To help understand how the spindle functions, a large amount of experimental data has been condensed into a simplified picture, in which the microtubules are divided into several categories (Fig. 2). First, one can divide the microtubules with respect to whether they end at the kinetochore or not. Kinetochore microtubules end at the kinetochore and form parallel bundles known as kinetochore fibers or k-fibers. Non-kinetochore microtubules can be found as single microtubules, in parallel bundles, or in antiparallel bundles. When divided with respect to their location, non-kinetochore microtubules include those that grow from the spindle pole towards the cell cortex, known as astral microtubules, those that grow Abstract When a cell starts to divide, it forms a spindle, a micro-machine made of microtubules, which separates the duplicated chromosomes. The attachment of microtubules to chromosomes is mediated by kinetochores, protein complexes on the chromosome. Spindle microtubules can be divided into three major classes: kinetochore microtubules, which form k-fibers ending at the kinetochore; interpolar microtubules, which extend from the opposite sides of the spindle and interact in the middle; and astral microtubules, which extend towards the cell cortex. Recent work in human cells has shown a close relationship between interpolar and kinetochore microtubules, where interpolar bundles are attached laterally to kinetochore fibers almost all along their length, acting as a bridge between sister k-fibers. Most of the interpolar bundles are attached to a pair of sister kinetochore fibers and vice versa. Thus, the spindle is made of modules consisting of a pair of sister kinetochore fibers and a bundle of interpolar microtubules that connects them. These interpolar bundles, termed bridging fibers, balance the forces acting at kinetochores and support the rounded shape of the spindle during metaphase. This review discusses the structure, function, and formation of kinetochore fibers and interpolar bundles, with an emphasis on how they interact. Their connections have an impact on the force balance in the spindle and on chromosome movement during mitosis because the forces in interpolar bundles are transmitted to kinetochore fibers and hence to kinetochores through these connections.
Winner of the EBSA Young Investigator Medal and Prize, 2015.
* Iva M. Tolić tolic@irb.hr towards the spindle equator and have free ends, which are often called polar, and those that form antiparallel overlaps in the central part of the spindle, known as interpolar or overlap microtubules (Alberts et al. 2014). This review is focused on kinetochore fibers and interpolar bundles, with an emphasis on the relationship between them in the context of human somatic cells. Even though these bundles are generally thought of as being physically separate in the region close to kinetochores, recent work suggests that they are tightly connected. This coupling has important implications for the forces acting on kinetochores and thus for the process of cell division.

Kinetochore fibers
Kinetochore fibers are the main generators of forces that move the chromosomes during mitosis. When sister kinetochores become attached to microtubule fibers extending from the opposite spindle poles, which is known as biorientation, these fibers hold the chromosomes near the spindle equator (Maiato et al. 2017). Their stable interaction with kinetochores is required for the cell to pass the spindle assembly checkpoint and proceed from metaphase into anaphase (Musacchio 2015). During anaphase, kinetochore fibers shorten by depolymerization at the kinetochore and at the pole, thereby segregating sister chromatids towards the opposite spindle poles (Asbury 2017).
Most of the existing information about kinetochore fiber structure has been obtained by electron microscopy studies (Fig. 3). The cross-section of a metaphase kinetochore fiber in PtK1 cells consists of 20-30 microtubules (McDonald et al. 1992;McEwen et al. 1997;Brinkley and Cartwright 1971;McIntosh et al. 1975). The number of microtubules in a kinetochore fiber increases as mitosis progresses and the fiber matures. At late prometaphase, the average number of microtubules on fully congressed kinetochores is 20, at late metaphase 24, and at early anaphase 28 (McEwen et al. 1997). In HeLa cells, a metaphase kinetochore fiber consists of 17 microtubules on average (McEwen et al. 2001;Wendell et al. 1993). The majority of the microtubules in a kinetochore fiber in PtK1 cells extend between the kinetochore and the spindle pole (Rieder 1981;McDonald et al. 1992;Brinkley and Cartwright 1971). The minus ends of these microtubules are found at the border of the spindle pole, roughly 0.4-0.5 µm away from the centrioles (McDonald et al. 1992).
Kinetochore fibers are the most stable bundles in the metaphase spindle, given that interpolar and astral microtubules disassemble after perturbations such as cold treatment, while kinetochore microtubules are more resistant (Brinkley and Cartwright 1975;Rieder 1981). The microtubules in a kinetochore fiber are linked by clathrin in a complex with the transforming acidic coiled-coil protein 3 (TACC3) and colonic, hepatic tumor overexpressed gene (ch-TOG). Clathrin is required for kinetochore fiber stability and proper chromosome congression to the metaphase plate (Booth et al. 2011;Royle et al. 2005). These proteins are part of a network of microtubule connectors, called the mesh (Nixon et al. 2015), which most likely pull kinetochore microtubules together and provide structural integrity to the kinetochore fiber.
During metaphase, kinetochore fibers show remarkable dynamic behavior. At the kinetochore end, they exhibit switching between growth and shrinkage, with a net growth over time Rieder and Salmon 1998). This growth is accompanied by the disassembly at the spindle pole, resulting in a flux of the entire kinetochore fiber poleward Mitchison 1989). Even though kinetochore fibers grow and shrink as a unit, the growth and shrinkage of individual microtubules in a single fiber is not necessarily synchronized. Electron tomography has revealed that plus ends of roughly 70% of kinetochore microtubules have curved protofilaments (VandenBeldt et al. 2006), suggesting that these microtubules are in a depolymerizing state based on in vitro observations that protofilaments of depolymerizing microtubules coil inside out (Mandelkow et al. 1991). Yet, this interpretation should be taken with caution because in vivo protofilaments can bend outwards even when the microtubule is growing (Kukulski et al. 2011;Hoog et al. 2007;McIntosh et al. 2013). Livecell imaging of EB3, a protein that tracks growing plus ends of microtubules, showed that a kinetochore fiber consists of a mixture of polymerizing and depolymerizing microtubules, with a small polymerization bias for fibers that exhibit net growth (Armond et al. 2015). Thus, the dynamics of individual microtubules within a kinetochore fiber is not coordinated, but this does not prevent the dynamic behavior of the fiber as a unit. How kinetochore fibers form in human cells is an open question. In principle, growth of the first microtubules of a nascent kinetochore fiber may be initiated at the spindle pole ( Fig. 4a) or at the kinetochore ( Fig. 4b) (Rieder 2005). If they grow from the spindle pole, they have to capture the kinetochores. A pioneering idea concerning the capture process is based on microtubule dynamics Holy and Leibler 1994). In this scenario, known as search-and-capture, microtubules grow in random directions from the spindle pole. If a microtubule does not interact with a kinetochore, it undergoes catastrophe and shrinks back to the pole. New microtubules grow, each of them having a chance to reach a kinetochore. A microtubule that eventually captures a kinetochore becomes stabilized by this interaction, thereby establishing the basis of a kinetochore fiber. Several mechanisms may accelerate this process (Wollman et al. 2005;Paul et al. 2009;Magidson et al. 2011Magidson et al. , 2015 and have been reviewed elsewhere (Mogilner and Craig 2010;Pavin and Tolic-Norrelykke 2014;Heald and Khodjakov 2015;Pavin and Tolic 2016;Prosser and Pelletier 2017).
Alternatively, growth of kinetochore microtubules may be initiated at the kinetochore (Fig. 4b). Microtubule growth from kinetochores has been seen on isolated human mitotic chromosomes (Telzer et al. 1975;McGill and Brinkley 1975) and in mammalian cells recovering from treatments with microtubule inhibitors (Witt et al. 1980;De Brabander et al. 1981). Initiation of kinetochore fiber formation at the kinetochore has also been observed in untreated cells (Khodjakov et al. 2003;Maiato et al. 2004). However, these events are rare and do not represent a dominant mechanism of kinetochore fiber formation in mammalian somatic cells.
A mature kinetochore fiber consists of about 20 parallel microtubules. This thick fiber is formed most likely by stepwise addition of new microtubules to the immature fiber. These new microtubules required for the maturation of the kinetochore fiber may grow from the spindle pole region along the existing fiber (Fig. 4c). A recent study supports this scenario by showing that kinesin-14 motors are recruited to the plus end of a microtubule and guide its growth along another microtubule (Molodtsov et al. 2016).
New microtubules growing from the spindle pole, which may eventually become part of an existing kinetochore fiber, do not necessarily grow along this fiber. Instead, they may grow at an angle with respect to the existing fiber, forming a V-shape with it (Fig. 4d). These V-shaped configurations are often seen in yeasts, where microtubules grow from the spindle pole body, a centrosome equivalent in yeast, in different directions (Sagolla et al. 2003). Interestingly, these microtubules rotate about the pivot at the spindle pole body (Kalinina et al. 2013;  Cojoc et al. 2016a). The concept of microtubule rotation, which is also known as pivoting, swiveling, or angular motion, may be important for the formation of kinetochore fibers because this motion helps the microtubules as they search for targets such as kinetochores, cortical anchors, or other microtubules (Pavin and Tolic-Norrelykke 2014). Pivoting is the dominant mechanism by which microtubules search for kinetochores during mitosis in fission yeast (Kalinina et al. 2013). Microtubule pivoting also contributes to kinetochore capture at the onset of meiosis I in the same organism (Cojoc et al. 2016a). Modeling work suggests that if the microtubules undergo both dynamic instability and rotation, the relative contribution of the dynamic instability versus rotation is higher for more dynamic microtubules (Cojoc et al. 2016a;Blackwell et al. 2017b). In Drosophila S2 cells, microtubules extending from the kinetochore are initially not oriented towards a spindle pole, but they pivot around the kinetochore while growing and eventually become captured by the microtubules growing from the spindle pole (Maiato et al. 2004). Microtubule pivoting also helps astral microtubules to find cortical anchor sites required for the movement of the spindle from the mother cell into the bud in budding yeast (Baumgartner and Tolic 2014). In all these cases, microtubule pivoting allows them to swipe through space, which increases the explored volume and makes the search process more efficient (Kalinina et al. 2013;Pavin and Tolic-Norrelykke 2014).
Pivoting of microtubules may be important for the transformation of a V-shaped microtubule structure into a parallel bundle, which may be relevant for the formation of kinetochore fibers (Fig. 4d). We have recently introduced a pivot-and-bond model, in which microtubules pivot around the spindle pole and bond with each other by crosslinking proteins (Prelogović et al. 2017). Our experiments in fission yeast show that microtubules pivot around the spindle pole before getting aligned and forming a parallel bundle, and that bundle formation relies to a large extent on the crosslinker anaphase spindle elongation protein (Ase1), a homolog of the human protein regulator of cytokinesis 1 (PRC1) (Prelogović et al. 2017). Thus, in the pivot-andbond model, microtubules explore the space by performing rotational diffusion and ultimately approach one another, which in turn allows the crosslinking proteins to connect the microtubules into a stable parallel bundle. Likewise, this mechanism may contribute to the formation and maturation of kinetochore fibers in human cells.
Finally, microtubules needed for the maturation of a kinetochore fiber may be formed at locations other than the spindle pole (Fig. 4e). For example, some microtubules are nucleated at sites along the preexisting mother microtubule  (a, b) and maturation (c-e) of kinetochore fibers. a, b Microtubules of the future kinetochore fiber are formed at the spindle pole or at the kinetochore, respectively. c New microtubules are formed at the pole and grow along the existing kinetochore microtubules. d New microtubules are formed at the pole and grow at an angle with respect to the existing microtubules, forming a V-shape. They rotate around the spindle pole and eventually approach the existing kinetochore microtubules, which is followed by their binding via crosslinking proteins (black spring). e New microtubules are nucleated at the nucleation sites (light blue) along the existing kinetochore fiber. In all panels, microtubules are shown as green lines, centrosomes as green circles with small cylinders representing centrioles inside, and chromosomes are purple with kinetochores depicted as dark purple circles a b d e Formation of a kinetochore fiber Growth from the pole Growth from the kinetochore Maturation of a kinetochore fiber Guided growth Rotation (pivoting) Nucleation along MT c (Murata et al. 2005;Goshima et al. 2008;Mahoney et al. 2006). In meiotic Xenopus egg extracts, these new microtubules grow at small angles and with the same polarity as the mother microtubule, which makes them suitable to generate parallel microtubule bundles such as kinetochore fibers (Petry et al. 2013). This mechanism may be at work also in human somatic cells.

Interpolar microtubule bundles
Interpolar bundles are important for the structural integrity of the spindle during its formation in prometaphase (Tanenbaum and Medema 2010). When the cell enters anaphase, interpolar bundles start to elongate, which results in pole separation and spindle elongation (Scholey et al. 2016). Because kinetochore fibers, which are attached to chromosomes, are mechanically connected to the pole either directly or indirectly via connections to other pole-bound microtubules, the separation of the spindle poles contributes to chromosome segregation.
As in the case of kinetochore fibers, the structure and spatial distribution of interpolar bundles has been revealed by electron microscopy (Fig. 5a). In PtK1 cells, the minus ends of interpolar microtubules are found throughout the spindle (Mastronarde et al. 1993), in contrast to the kinetochore microtubules whose minus ends are mostly near the poles (McDonald et al. 1992). Interpolar microtubules meet in the equatorial region of the spindle, where they form antiparallel overlaps (Brinkley and Cartwright 1971). In addition, a substantial fraction of non-kinetochore microtubules end before they reach the spindle equator (McIntosh et al. 1975).
The major passive crosslinker of antiparallel interpolar microtubules is PRC1/Ase1 (Pellman et al. 1995;Mollinari et al. 2002;Bieling et al. 2010;Jiang et al. 1998). Thus, PRC1 can be used to visualize the overlap zones of interpolar microtubules throughout the spindle (Fig. 5b). During metaphase, both endogenous and GFP-tagged PRC1 is found in the central part of the spindle, in streaks running roughly parallel to the spindle axis (Polak et al. 2017). The PRC1 streaks are about 5 µm long, reflecting the length of the antiparallel overlaps. Electron micrographs revealed overlap zones of a similar length (Mastronarde et al. 1993). These overlaps become shorter in late anaphase, which is visible both in electron micrographs (Mastronarde et al. 1993) and as accumulation of PRC1 in short bands in the spindle midzone (Jiang et al. 1998;Mollinari et al. 2002).
Microtubules that form antiparallel overlaps can slide with respect to one another. Early studies on diatom and fission yeast spindles have shown that sliding of interpolar microtubules, which is powered by motor proteins, is the key mechanochemical process driving anaphase spindle elongation (Cande and McDonald 1985;Masuda et al. 1990). For metaphase spindles, early work on budding yeast has established that counteracting forces produced by oppositely oriented motors maintain the spindle structure (Saunders and Hoyt 1992). In particular, kinesin-5 motors Cut7/Cin8/Eg5/KIF11 slide the microtubules, and thus the spindle poles, apart by walking along the microtubules away from the pole, i.e., towards the plus end of the microtubules (Hagan and Yanagida 1990;Le Guellec et al. 1991;Sawin et al. 1992;Kapitein et al. 2005;Saunders and Hoyt 1992). Other motors pull the spindle poles towards each other by walking along the microtubules towards the pole, i.e., towards the minus end of the microtubule, such as kinesin-14 motors Ncd/HSET/KifC1 (Endow et al. 1990;McDonald et al. 1990;Cai et al. 2009) a b * * Fig. 5 Interpolar bundles. a Interpolar microtubules in a PtK1 cell in early anaphase, reconstructed from an electron micrograph. Microtubules whose minus ends were associated with the pole on the left or the right are shown separately. Scale bar 1 µm. Image reproduced with permission from (Mastronarde et al. 1993). b Interpolar bundles in a HeLa cell visualized by PRC1-GFP (green), which binds to antiparallel microtubule overlaps. The antiparallel overlap zones are ~5 µm long and found in the central part of the spindle. Kinetochores (mRFP-CENP-B) are visible in magenta. Asterisks mark the spindle poles; scale bar 1 µm. Image reproduced with permission from (Polak et al. 2017) and dynein (Tanenbaum et al. 2008(Tanenbaum et al. , 2013. Interestingly, kinesin-5 motors can also move towards the minus end of the microtubules, when walking on a single microtubule or in a non-crowded environment on antiparallel microtubules (Roostalu et al. 2011;Edamatsu 2014;Britto et al. 2016). Similarly, kinesin-14 can reverse its direction of movement from the minus end toward the plus end under a low external force (Molodtsov et al. 2016). Coordination of the forces exerted by motor proteins, together with the forces arising from microtubule dynamics, are thought to be responsible for the maintenance of a constant spindle length during metaphase and for spindle elongation in anaphase (Brust-Mascher et al. 2004;Goshima and Scholey 2010;Sharp et al. 1999;Scholey et al. 2016;Saunders and Hoyt 1992).
As in the case of kinetochore fibers, the process of formation of interpolar bundles in human cells is not well understood. Presumably, interpolar bundles form during early mitosis through the interactions of microtubules growing from the opposite spindle poles. In the search-and-capture picture ), as discussed above, microtubules grow in random directions from the spindle poles (Fig. 6a). Because of this dynamic behavior, microtubules sometimes get close to those extending from the other spindle pole. In the region where such microtubules meet, they become crosslinked by specific crosslinkers and motor proteins, which stabilizes the microtubules against shrinking. Formation of interpolar bundles through these processes has been explored by computer simulations (Nédélec 2002).
The swiping motion of microtubules during their rotation around the spindle pole, which was found in yeasts (Kalinina et al. 2013;Baumgartner and Tolic 2014;Cojoc et al. 2016a;Pavin and Tolic-Norrelykke 2014), may help the microtubules extending from the two poles to find each other much faster than in the case without rotation (Fig. 6b). A recent study incorporated microtubule rotation around the spindle pole body into a model of spindle formation in fission yeast (Blackwell et al. 2017a). Computer simulations of that model suggest that a decreased microtubule rotation results in shorter spindles and fewer microtubules in the bundle connecting the two spindle poles. Thus, proper spindle assembly requires the connections between the microtubules and the spindle pole body to be strong to keep them linked and at the same time flexible to allow free rotation of microtubules.
Microtubule rotation may occur not only before the microtubules from the opposite spindle poles meet, but also after their initial interaction. Indeed, during the formation of interpolar bundles, microtubules that meet at an oblique angle must rotate to get aligned in an antiparallel fashion. A study of spindle assembly in budding yeast showed that cells lacking kinesin-14 motors have poorly aligned interpolar microtubules (Hepperla et al. 2014). This finding was explained by a model in which microtubules growing from the two spindle poles become connected by kinesin-14 motors. The motors walk along one microtubule towards its minus end, while being bound to other microtubule, thereby aligning the two microtubules into an interpolar bundle (Fig. 6c). Thus, in this model, Microtubules rotate around the spindle pole (arrows), which allows them to explore the space more efficiently and approach the microtubules extending from the opposite pole more quickly. c Microtubules extending from the opposite poles are connected by minus end directed motors (small black cherry-like object), which align the two microtubules (curved arrow) as they walk towards the spindle pole (straight arrow). In all panels, microtubules are shown as green lines, and centrosomes as green circles with small cylinders representing centrioles motor-driven angular motion of microtubules results in their alignment and formation of an antiparallel bundle (Hepperla et al. 2014). Even though the concept of angular motion of microtubules comes from studies on yeast cells, it may also be relevant for the formation of interpolar bundles in human cells.

Connections between kinetochore fibers and interpolar microtubules
A textbook picture of the spindle depicts kinetochore fibers and interpolar bundles as distinct clusters, physically separated from each other along their length except in the regions near the spindle poles (Fig. 2). The spindle is evidently simplified in this picture, lacking a large part of the complex spatial distribution and intricate connections between its building blocks. Nevertheless, the current view in the field is that kinetochore fibers and interpolar bundles are largely independent structures in the region close to kinetochores.
Remarkably, several studies on cells from various species have shown that different microtubule bundles in the spindle are in close contact. Electron micrographs have revealed non-kinetochore microtubules that extend along the kinetochore fiber, pass the kinetochore and enter the region between sister kinetochores, in metaphase spindles of human WI-38 cells (Fig. 7a) (McIntosh and Landis 1971) and HeLa cells (Nixon et al. 2017), plant endosperm (Fig. 7b) (Jensen 1982), and Xenopus egg extracts (Fig. 7c) (Ohi et al. 2003). Intermixing of nonkinetochore microtubules with kinetochore fibers has been seen in the region close to the kinetochore in PtK1 cells (Fig. 7d) Fig. 7 Interpolar microtubules are found in the vicinity of kinetochore microtubules and kinetochores during metaphase. Example images of metaphase spindles from different organisms, obtained by using electron microscopy, are shown. a Area around a kinetochore in a human WI-38 cell. Several microtubules end at the kinetochore, whereas a non-kinetochore microtubule passes the kinetochore zone. Image reproduced with permission from (McIntosh and Landis 1971). b Area around kinetochores in the spindle of Haemanthus katherinae endosperm. Two kinetochores (K 1 and K 2 ) are attached to kinetochore microtubules (K). Non-kinetochore microtubules (long arrows) intermingle with kinetochore microtubules. Image reproduced with permission from (Jensen 1982). c Area around kinetochores in a Xenopus egg extract spindle. Kinetochore fibers are asso-ciated with microtubules that do not end at kinetochores (arrowhead). Image reproduced with permission from (Ohi et al. 2003). d Area around kinetochores in a PtK1 cell. Interpolar microtubules (IMT) and microtubules which penetrate the chromosome (white arrow) are found near the kinetochores (K). Magnification ×21,000; image reproduced with permission from (Brinkley and Cartwright 1971). e Arrangement of interpolar and kinetochore microtubules in a metaphase PtK1 spindle, based on reconstructions of microtubules from electron micrographs. Black circles mark the minus ends of interpolar microtubules, which are found farther from the pole than those of kinetochore microtubules and most are in or near kinetochore fibers. Scale bar 1 µm; image reproduced with permission from (Mastronarde et al. 1993) 1971). The minus ends of interpolar microtubules were found to be clustered in the bundles of kinetochore microtubules (Fig. 7e) (Mastronarde et al. 1993). In fission yeast and budding yeast, interpolar microtubules form a single bundle, while kinetochore microtubules lie next to it Winey et al. 1995). Thus, electron microscopy has revealed that non-kinetochore microtubules can be found in the neighborhood of kinetochore microtubules in various cell types, which implies that the microtubules from these two groups may be physically linked. The existence of such links may be important for the force balance in the spindle and chromosome movement because the forces generated by non-kinetochore microtubules, in particular by the interpolar bundles, may be transmitted to kinetochore fibers and hence to kinetochores through these connections.
Connections between different fibers in the metaphase spindle have been included in physical models. In one model, kinetochore fibers and interpolar microtubules are coupled by viscoelastic links (Matos et al. 2009). This model suggests that the poleward flux of spindle microtubules is able to equalize the tension over all kinetochores, ensuring coordinated segregation of chromosomes in anaphase (Pereira and Maiato 2012;Matos et al. 2009). In another model, neighboring kinetochore fibers are connected by elastic springs. This model was used to explain the observation that neighboring kinetochore pairs oscillate in a coordinated manner (Vladimirou et al. 2013). Thus, both models indicate that connections between neighboring fibers can synchronize the dynamics of different kinetochore pairs during metaphase.
In anaphase, the current understanding of forces acting on chromosomes is based on events occurring at the ends of the kinetochore fiber. These forces are associated with shortening of kinetochore microtubules at the kinetochore and at the spindle pole (Maiato and Lince-Faria 2010;Asbury 2017). In addition, forces can be generated at the kinetochore fiber end created by laser cutting, where dynein is recruited and drives poleward movement of the end of the kinetochore fiber by walking along neighboring pole-anchored microtubules (Elting et al. 2014;Sikirzhytski et al. 2014). Yet, early studies suggested that forces act along the length of the kinetochore fiber rather than only at its ends (Ostergren 1951). These forces may be generated by the sliding of antiparallel interpolar microtubules over one another (McIntosh et al. 1969) and transmitted to kinetochore fibers through the crosslinks between interpolar and kinetochore microtubules (Margolis et al. 1978;Goode 1981;Mitchison 2005). Thus, forces acting along the lattice of kinetochore fiber microtubules may contribute to the movement of chromosomes in anaphase. However, these ideas have not been directly tested so far.

Interpolar microtubules act as a bridge between sister kinetochore fibers
We have recently explored the relationship between interpolar and kinetochore fibers (Kajtez et al. 2016). By using fluorescence microscopy images of live human cells in metaphase, we observed that a bundle of interpolar microtubules connects a pair of kinetochore fibers bound to sister kinetochores (Fig. 8a). This interpolar bundle looks like a bridge between sister kinetochore fibers, hence we called it bridging fiber. It is important to note that the bridging fiber is defined by its function, as a fiber that links two sister kinetochore fibers.
The non-kinetochore microtubules found in the vicinity of kinetochores, illustrated in Fig. 7, may be bridging microtubules. However, images such as those shown in Figs. 7 and 8a cannot distinguish whether these microtubules are physically linked with kinetochore fibers or they just happened to lie close to them. Thus, to test the interaction between different microtubules in the spindle, it was crucial to develop an assay that can identify such interactions. We used laser ablation to sever a kinetochore fiber in HeLa, U2OS and PtK1 cells (Buda et al. 2017;Kajtez et al. 2016;Milas and Tolic 2016), similar to previous cutting experiments (Cojoc et al. 2016b;Elting et al. 2014;Sikirzhytski et al. 2014). We reasoned that if the bridging fiber is physically linked with kinetochore fibers, they are expected to move together after the severing. Indeed, we observed that the bridging fiber moved together with sister kinetochores, the kinetochore fiber stub that remained attached to the kinetochore after the severing, and the intact kinetochore fiber of the sister kinetochore ( Fig. 8b) (Kajtez et al. 2016). All these structures moved as a single object away from the spindle, and later back towards the spindle. Thus, the bridging fiber is indeed strongly linked to kinetochore fibers, acting as a bridge between them.
Severing of a kinetochore fiber at different locations revealed that the kinetochore fiber is laterally linked with the bridging fiber in a large region starting ~1 µm away from the kinetochore and extending towards the spindle pole Kajtez et al. 2016). In the region up to ~1 µm away from the kinetochore, the kinetochore fiber and the bridging fiber are separated, with a distance between them of ~250 nm at the location of the kinetochore (Kajtez et al. 2016).
The bridging fiber consists of 10-15 microtubules arranged in an anti-parallel manner, based on the observation that they bind PRC1 (Fig. 8c), a crosslinking protein localized in the antiparallel overlaps of microtubules in vitro (Bieling et al. 2010;Subramanian et al. 2013;Kapitein et al. 2008) and of the spindle midzone (Jiang et al. 1998;Mollinari et al. 2002;Pellman et al. 1995). By taking advantage of the variable karyotype of HeLa cells, we found that the number of PRC1-decorated bundles per spindle roughly matches the number of kinetochore pairs, indicating a nearly one-to-one relationship between the interpolar bundles and chromosomes (Polak et al. 2017). Localization of the PRC1-labeled bundles with respect to kinetochores showed that more than 90% of PRC1 bundles are associated with a pair of sister kinetochores. Thus, virtually all interpolar bundles in a metaphase spindle are bridging fibers (Fig. 9). In other words, there are practically no "free" overlap bundles in an unperturbed metaphase spindle.
Similarly, more than 90% of kinetochore pairs in a spindle have a bridging fiber connecting their kinetochore fibers in metaphase (Polak et al. 2017). This observation argues against an alternative interpretation in which the non-kinetochore microtubules lying between sister kinetochores are a result of merotelic attachments, where one sister kinetochore is attached to microtubules emanating from both spindle poles (Gregan et al. 2011).
It is still unknown where the plus and minus ends of the bridging microtubules are located. The PRC1-decorated overlap zone of antiparallel microtubules extends roughly 2 µm along each sister k-fiber away from the kinetochore (Kajtez et al. 2016;Polak et al. 2017), which indicates that Bridging microtubules link sister kinetochore fibers. a Spindle in a HeLa cell with microtubules shown in green (stained with SiR-tubulin) and kinetochores in magenta (EGFP-CENP-A). Enlargements of the boxed region (top tubulin, bottom merge) show bridging fibers connecting sister kinetochore fibers (k-fibers). Compared with kinetochore fibers, bridging fibers contain fewer microtubules and hence are less bright. b Laser ablation of a kinetochore fiber in a HeLa cell with microtubules shown in green (tubulin-GFP) and kinetochores in magenta (mRFP-CENP-B). Time-lapse images of the spindle (top) and enlargements of the boxed region (middle tubulin, bottom schemes) are shown. After the cut (yellow lightning sign), the bridging fiber moved together with sister kinetochores and their fibers in the direction away from the spindle. Image reproduced with permission from (Kajtez et al. 2016). c Spindle in a HeLa cell with microtubules shown in green (tubulin-GFP) and endogenous PRC1 in magenta (immunostained, Alexa Fluor-555 labeled). Enlargements of the boxed region (top merge, middle: PRC1, bottom tubulin). The PRC1 signal is found in the central part of the bridging fiber, extending ~2 µm poleward from each kinetochore. Image reproduced with permission from (Polak et al. 2017). Scale bars in all panels are 1 µm Bridging microtubules Kinetochore microtubules Fig. 9 Revised scheme of the spindle. The spindle in a human somatic cell is made of modules consisting of a pair of sister kinetochore fibers and a bridging fiber that connects them. Kinetochore microtubules are shown in purple and all non-kinetochore ones, including bridging microtubules, in green. Centrosomes are indicated as green circles with small cylinders representing centrioles, and chromosomes are purple with kinetochores depicted as dark purple circles the plus ends of bridging microtubules are found within that region. Previous electron microscopy experiments have shown that the minus ends of interpolar microtubules are found along kinetochore fibers (Fig. 7e) (Mastronarde et al. 1993). Thus, the minus ends of bridging microtubules are likely found along the kinetochore fibers (Fig. 9). Identification of the sites where the plus and minus ends of bridging microtubules are situated will require further work.

The bridging fiber balances the tension between sister kinetochores
In addition to the experimental characterization of the microtubules in the spindle, physical models are necessary for quantitative understanding of forces acting in the spindle (Tolic-Norrelykke 2008;Dumont and Mitchison 2009;Tolic-Norrelykke 2010). In most models, the physical linkage between sister kinetochores is described as an elastic connection, which is mediated by centromeric chromatin (Bloom and Joglekar 2010;Burrack and Berman 2012). Thus, a pair of sister kinetochore fibers and their kinetochores are typically represented by two rods connected by an elastic spring (Joglekar and Hunt 2002;Civelekoglu-Scholey et al. 2013;Vladimirou et al. 2013).
We have recently introduced a physical model that includes the bridging fiber as a link between sister kinetochore fibers (Fig. 10) (Kajtez et al. 2016). In this model, kinetochore fibers and the bridging fiber are elastic rods, which bend under compression. From the observed curvature of the fibers, we assessed the forces acting on them. Our model suggests that the observed shape of the outermost kinetochore fibers can be obtained if a compressive force of ~50 pN acts at the pole, tension of ~300 pN at the kinetochore, and the junction point where the kinetochore fiber merges with the bridging fiber is located ~1 µm away from the kinetochore.
Interestingly, we found that tension and compression coexist along the same kinetochore fiber (Fig. 10) (Kajtez et al. 2016;Tolic and Pavin 2016). The kinetochore fiber is under tension in the region between the kinetochore and the junction point. Conversely, the kinetochore fiber is under compression along the largest part of its length, where the kinetochore fiber is laterally linked with the bridging fiber, i.e., between the junction and the spindle pole. Thus, our model resolves the paradox of the simultaneous existence of tension and compression along a single kinetochore fiber, discussed in (Dumont and Mitchison 2009), by suggesting that the compression in the bridging fiber balances the tension between sister kinetochores and the compression at the spindle pole.

Open questions
The work on the role of the bridging fibers has opened numerous questions. To what extent is the concept of bridging fibers relevant for other organisms? The findings described here suggest that in mammalian cells the spindle is made of modules consisting of a pair of sister kinetochore fibers and a bundle of interpolar microtubules that connects them. In yeasts, the whole spindle may be thought of as being equivalent to an individual module from a mammalian spindle. Yet, this idea remains to be tested.
How and when is the bridge between sister kinetochore fibers built? In one scenario, kinetochores interact first with an overlap bundle, which will become the bridging fiber after the kinetochore fibers are formed. In the other scenario, kinetochore fibers form first and the bridging fiber is subsequently acquired (Simunic and Tolic 2016). What is the role of the bridging fiber in spindle assembly? Depending on which of the two mentioned scenarios is more applicable to human cells, the bridging fiber may contribute to different extent to the formation of the spindle, congression of the chromosomes to the metaphase plate, establishment of biorientation, setting of the spindle length and maintenance of its structural integrity.
What happens with the bridging fiber during anaphase? If it remains intact and attached to kinetochore fibers, it may contribute to the separation of sister kinetochores. This fiber may serve as a mechanical support for the kinetochore fibers as they shorten and move the kinetochores poleward. Moreover, motor proteins may generate sliding of kinetochore fibers poleward along the bridging fiber. Similarly, microtubules in the bridging fiber may slide apart, pushing the attached kinetochore fibers poleward. It will be exciting to examine these scenarios, both experimentally and theoretically.
Future work will reveal the role of the bridging fiber in critical stages of mitosis including spindle assembly Tension Compression Kinetochore Bridging fiber Kinetochore fiber Fig. 10 Force balance based on a theoretical model that includes a bridging fiber as a link between sister kinetochore fibers. The compression in the bridging fiber balances the tension on kinetochores and the compression at the spindle pole (arrows represent forces). Thus, the bridging fiber allows existence of tension and compression (purple and green segments, respectively) within an individual kinetochore fiber (Kajtez et al. 2016) and chromosome segregation. The forces resulting from crosslinking kinetochore fibers and interpolar bundles may emerge as an important part of mitosis.