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Cells fine-tune microtubule assembly in both space and time, to give rise to distinct edifices with specific cellular functions. In proliferating cells, microtubules are highly dynamics, yet, proliferation cessation often lead to their stabilization. One of the most stable microtubule structures identified to date is the nuclear bundle assembled in yeast quiescent cells. In this report, we characterize the original multistep process driving the assembly of this structure. We show that its follows a precise temporality that relies on the sequential action of specific kinesin-14/kinesins-5 and involves both microtubule-kinetochore and kinetochore-kinetochore interactions. Upon quiescence exit, the microtubule bundle disassembles via a cooperative process involving the Kinesin-8 and its full disassembly is required to authorize cells re-entry into proliferation. Overall, our study not only provides the first description, at the molecular scale, of the entire life cycle of a stable microtubule structurein vivo, but also sheds light on its function as a sort of “checkpoint” for cell cycle resumption.

Mitochondria play a crucial role in most eukaryotic cells. Mitophagy is a process that controls their quality and quantity within the cells. The outer mitochondrial membrane protein, Atg32, serves as the mitophagic receptor. It interacts with the Atg11 protein to initiate mitophagy and with the Atg8 protein to ensure the engulfment of mitochondria into the autophagosomes for elimination. The Atg32 protein is regulated at the transcriptional level but also by posttranslational modifications. In this study, we described a new regulator of mitophagy, the protein Dep1, identified as a part of the Rpd3L histone deacetylase complex. We showed that the Dep1 protein is localized in the nucleus and associated with mitochondria. This protein is needed for mitophagy and to regulate the transcription and expression of the Atg32 protein. The absence of this protein affects the mitophagy process induced by either starvation for nitrogen or the stationary phase of growth.

Mitochondrial ATP-synthesis is catalyzed by a F1Fo-ATP synthase, an enzyme of dual genetic origin enriched at the edge of cristae where it plays a key role in their structure/stability. The enzyme’s biogenesis remains poorly understood, both from a mechanistic and a compartmentalization point of view. The present study provides novel molecular insights into this process through investigations on a human protein called TMEM70 with an unclear role in the assembly of ATP synthase. A recent study has revealed the existence of physical interactions between TMEM70 and the subunit c (Su.c), a protein present in 8 identical copies forming a transmembrane oligomeric ring (c-ring) within the ATP synthase proton translocating domain (FO). Herein we analyzed the ATP-synthase assembly in cells lacking TMEM70, mitochondrial DNA or F1 subunits and observe a reciprocal dependence of TMEM70 and Su.c levels, regardless of the status of other ATP synthase subunits or of mitochondrial bioenergetics. Immunoprecipitation and two-dimensional blue-native/SDS-PAGE reveal that TMEM70 forms large oligomers composed of 8 TMEM70 dimers and that TMEM70 oligomers interact with Su.c not yet incorporated into ATP synthase complexes. Moreover, discrete TMEM70-Su.c complexes with increasing Su.c contents can be detected, suggesting a role for TMEM70 oligomers in the gradual assembly of the c-ring. Furthermore, we demonstrate using expansion super-resolution microscopy the specific localization of TMEM70 at the inner cristae membrane, distinct from the MICOS component MIC60. Taken together, our results show that TMEM70 oligomers provide a scaffold for c-ring assembly and that mammalian ATP synthase is assembled within inner cristae membranes.

Identifying synthetic lethal interactions has emerged as a promising new therapeutic approach aimed at targeting cancer cells directly. Here, we used the yeast Saccharomyces cerevisiae as a simple eukaryotic model to screen for mutations resulting in a synthetic lethality with 5-amino-4-imidazole carboxamide ribonucleoside (AICAR) treatment. Indeed, AICAR has been reported to inhibit the proliferation of multiple cancer cell lines. Here, we found that loss of several histone-modifying enzymes, including Bre1 (histone H2B ubiquitination) and Set1 (histone H3 lysine 4 methylation), greatly enhanced AICAR inhibition on growth via the combined effects of both the drug and mutations on G1 cyclins. Our results point to AICAR impacting on Cln3 subcellular localization and at the Cln1 protein level, while the bre1 or set1 deletion affected CLN1 and CLN2 expression. As a consequence, AICAR and bre1/set1 deletions jointly affected all three G1 cyclins (Cln1, Cln2, and Cln3), leading to a condition known to result in synthetic lethality. Significantly, these chemo-genetic synthetic interactions were conserved in human HCT116 cells. Indeed, knock-down of RNF40, ASH2L, and KMT2D/MLL2 induced a highly significant increase in AICAR sensitivity. Given that KMT2D/MLL2 is mutated at high frequency in a variety of cancers, this synthetic lethal interaction has an interesting therapeutic potential.

International audience; AICAR (Acadesine) is a pharmacological precursor of purine nucleotide biosynthesis with anti-tumoral properties. Although recognized as an AMP mimetic activator of the protein kinase AMPK, the AICAR monophosphate derivative ZMP was also shown to mediate AMPK-independent effects. In order to unveil these AMPK-independent functions, we performed a transcriptomic analysis in AMPKα1/α2 double knockout murine embryonic cells. Kinetic analysis of the cellular response to AICAR revealed the up-regulation of the large tumor suppressor kinases (Lats) 1 and 2 transcripts, followed by the repression of numerous genes downstream of the transcriptional regulators Yap1 and Taz. This transcriptional signature, together with the observation of increased levels in phosphorylation of Lats1 and Yap1 proteins, suggested that the Hippo signaling pathway was activated by AICAR. This effect was observed in both fibroblasts and epithelial cells. Knockdown of Lats1/2 prevented the cytoplasmic delocalization of Yap1/Taz proteins in response to AICAR and conferred a higher resistance to the drug. These results indicate that activation of the most downstream steps of the Hippo cascade participates to the antiproliferative effects of AICAR.

AICAR (Acadesine) is a pharmacological precursor of purine nucleotide biosynthesis with anti-tumoral properties. Although recognized as an AMP mimetic activator of the protein kinase AMPK, the AICAR monophosphate derivative ZMP was also shown to mediate AMPK-independent effects. In order to unveil these AMPK-independent functions, we performed a transcriptomic analysis in AMPKα1/α2 double knockout murine embryonic cells. Kinetic analysis of the cellular response to AICAR revealed the up-regulation of the large tumor suppressor kinases (Lats) 1 and 2 transcripts, followed by the repression of numerous genes downstream of the transcriptional regulators Yap1 and Taz. This transcriptional signature, together with the observation of increased levels in phosphorylation of Lats1 and Yap1 proteins, suggested that the Hippo signaling pathway was activated by AICAR. This effect was observed in both fibroblasts and epithelial cells. Knockdown of Lats1/2 prevented the cytoplasmic delocalization of Yap1/Taz proteins in response to AICAR and conferred a higher resistance to the drug. These results indicate that activation of the most downstream steps of the Hippo cascade participates to the antiproliferative effects of AICAR.

Microtubules are involved in plant development and adaptation to their environment, but the sustaining molecular mechanisms remain elusive. Microtubule-end-binding 1 (EB1) proteins participate in directional root growth in Arabidopsis thaliana. However, a connection to the underlying microtubule array has not been established yet. We show here that EB1 proteins contribute to the organization of cortical microtubules in growing epidermal plant cells, without significant modulation of microtubule dynamics. Using super-resolution stimulated emission depletion (STED) microscopy and an original quantification approach, we also demonstrate a significant reduction of apparent microtubule bundling in cytoplasmic-EB1-deficient plants, suggesting a function for EB1 in the interaction between adjacent microtubules. Furthermore, we observed root growth defects in EB1-deficient plants, which are not related to cell division impairment. Altogether, our results support a role for EB1 proteins in root development, in part by maintaining the organization of cortical microtubules. This article has an associated First Person interview with the first author of the paper.

The fission yeast cytoskeleton is completely reorganized upon quiescence entry: the microtubule (MT) cytoskeleton is drastically reshaped as quiescent cells assemble a large spindle pole body–associated MT bundle composed of stable antiparallel MTs., Cells perpetually face the decision to proliferate or to stay quiescent. Here we show that upon quiescence establishment, Schizosaccharomyces pombe cells drastically rearrange both their actin and microtubule (MT) cytoskeletons and lose their polarity. Indeed, while polarity markers are lost from cell extremities, actin patches and cables are reorganized into actin bodies, which are stable actin filament–containing structures. Astonishingly, MTs are also stabilized and rearranged into a novel antiparallel bundle associated with the spindle pole body, named Q-MT bundle. We have identified proteins involved in this process and propose a molecular model for Q-MT bundle formation. Finally and importantly, we reveal that Q-MT bundle elongation is involved in polarity reestablishment upon quiescence exit and thereby the efficient return to the proliferative state. Our work demonstrates that quiescent S. pombe cells assemble specific cytoskeleton structures that improve the swiftness of the transition back to proliferation.

CLIP-170 is a plus-end tracking protein which may act as an anticatastrophe factor. It has been proposed to mediate the association of dynein/dynactin to microtubule (MT) plus ends, and it also binds to kinetochores in a dynein/dynactin-dependent fashion, both via its C-terminal domain. This domain contains two zinc finger motifs (proximal and distal), which are hypothesized to mediate protein-protein interactions. LIS1, a protein implicated in brain development, acts in several processes mediated by the dynein/dynactin pathway by interacting with dynein and other proteins. Here we demonstrate colocalization and direct interaction between CLIP-170 and LIS1. In mammalian cells, LIS1 recruitment to kinetochores is dynein/dynactin dependent, and recruitment there of CLIP-170 is dependent on its site of binding to LIS1, located in the distal zinc finger motif. Overexpression of CLIP-170 results in a zinc finger-dependent localization of a phospho-LIS1 isoform and dynactin to MT bundles, raising the possibility that CLIP-170 and LIS1 regulate dynein/dynactin binding to MTs. This work suggests that LIS1 is a regulated adapter between CLIP-170 and cytoplasmic dynein at sites involved in cargo-MT loading, and/or in the control of MT dynamics.

International audience; Cell fusion occurs as part of the differentiation of some cell types, including myotubes in muscle and osteoclasts in remodeling bone. In the human placenta, mononuclear cytotrophoblasts in a human chorionic gonadotropin (hCG)-driven process fuse to form multinucleated syncytia that allow the exchange of nutrients and gases between the maternal and fetal circulation. Experiments in which protein kinase A (PKA) is displaced from A-kinase anchoring proteins (AKAPs), or in which specific AKAPs are depleted by siRNA-mediated knockdown, point to ezrin as a scaffold required for hCG-, cAMP-and PKA-mediated regulation of the fusion process. By a variety of immunoprecipitation and immunolocalization experiments, we show that ezrin directs PKA to a molecular complex of connexin 43 (Cx43, also known as GJA1) and zona occludens-1 (ZO-1, also known as TJP1). A combination of knockdown experiments and reconstitution with ezrin or Cx43 with or without the ability to bind to its interaction partner or to PKA demonstrate that ezrin-mediated coordination of the localization of PKA and Cx43 is necessary for discrete control of Cx43 phosphorylation and hCG-stimulated gap junction communication that triggers cell fusion in cytotrophoblasts.

The mitotic spindle is often positioned in a characteristic location during development, for example to enable the proper segregation of developmental determinants [1,2]. When epithelial cells divide, the mitotic spindle is often positioned parallel to the plane of the epithelium, so that both daughter cells contribute to the epithelium [3]. The mechanisms by which mitotic spindles are positioned have not been characterized in great detail, but evidence is accumulating that in some systems the dynein–dynactin microtubule motor complex plays a role [4–6]. Dynein has yet not been localized to cortical sites where it could bind to microtubules and exert a force that might orient the mitotic spindle, however [7,8]. Here, we report that in mitotic polarized epithelial cells, the dynein–dynactin complex accumulates, from prometaphase onwards, along astral microtubules and at cortical spots, into which many of the astral microtubules dock. The spots are assembled at the lateral plasma membrane, in the region below the tight junctions. Their formation is inhibited by cytochalasin D, and under these conditions the spindles do not orient properly. This novel localization of the dynein–dynactin complex is consistent with a role for the complex in the positioning of the mitotic spindle. We also show that, during prophase, the motor complex colocalizes with the nuclear envelope, consistent with it having a role in separating the centrosomes that are associated with the nuclear envelope.

International audience; Connexins (Cx) are key regulators of cell proliferation, differentiation and apoptosis. Cx trafficking and endocytosis need interactions with a large number of signaling and scaffolding proteins. We demonstrate herein that Cx43-GFP gap junction plaque endocytosis was blocked in cells transfected by the dominant-negative form of dynamin2 (Dyn2K44A) and by dynasore, an inhibitor of dynamin GTPase activity, which reduced the association between dynamin2 and Cx43. Our data also reveal that recruitment of the GTPase at the plasma membrane and its activation by c-Src are key events for Cx43 internalization. In addition they show that dynamin2 participated in internalization and degradation of the gap junction plaque but also in recycling of Cx43 to the plasma membrane through respectively Rab5/Rab7 and Rab11 pathways. These results demonstrate for the first time that dynamin2 is a new Cx partner and report an innovating mechanistic model by which dynamin2 may control Cx43 gap junction plaque invagination, endocytosis, recycling and degradation. These processes are magnified in response to carcinogen exposure underlining their potential importance during carcinogenesis.

International audience; In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, beta-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO(-/-)) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO(-/-) as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO(-/-) testis. Occludin, N-cadherin and beta-catenin levels were enhanced in SCCx43KO(-/-) mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.

A defect in microtubule (MT)-based transport contributes to the neuronal toxicity observed in Huntington's disease (HD). Histone deacetylase (HDAC) inhibitors show neuroprotective effects in this devastating neurodegenerative disorder. We report here that HDAC inhibitors, including trichostatin A (TSA), increase vesicular transport of brain-derived neurotrophic factor (BDNF) by inhibiting HDAC6, thereby increasing acetylation at lysine 40 of α-tubulin. MT acetylationin vitroand in cells causes the recruitment of the molecular motors dynein and kinesin-1 to MTs. In neurons, acetylation at lysine 40 of α-tubulin increases the flux of vesicles and the subsequent release of BDNF. We show that tubulin acetylation is reduced in HD brains and that TSA compensates for the transport- and release-defect phenotypes that are observed in disease. Our findings reveal that HDAC6 inhibition and acetylation at lysine 40 of α-tubulin may be therapeutic targets of interest in disorders such as HD in which intracellular transport is altered.

Polyglutamine expansion (polyQ) in the protein huntingtin is pathogenic and responsible for the neuronal toxicity associated with Huntington's disease (HD). Although wild-type huntingtin possesses antiapoptotic properties, the relationship between the neuroprotective functions of huntingtin and pathogenesis of HD remains unclear. Here, we show that huntingtin specifically enhances vesicular transport of brain-derived neurotrophic factor (BDNF) along microtubules. Huntingtin-mediated transport involves huntingtin-associated protein-1 (HAP1) and the p150Glued subunit of dynactin, an essential component of molecular motors. BDNF transport is attenuated both in the disease context and by reducing the levels of wild-type huntingtin. The alteration of the huntingtin/HAP1/p150Glued complex correlates with reduced association of motor proteins with microtubules. Finally, we find that the polyQ-huntingtin-induced transport deficit results in the loss of neurotrophic support and neuronal toxicity. Our findings indicate that a key role of huntingtin is to promote BDNF transport and suggest that loss of this function might contribute to pathogenesis.

Connexins form gap junction channels that allow intercellular communication between neighboring cells. Compelling evidence has revealed that Cx are tumor-suppressor genes and reduced Cx expression has been related with uncontrolled cell growth in tumors and transformed cells. In the present study, we addressed Cx transcriptional and posttranscriptional regulations during the earlier stage of testicular tumors confined to Leydig cells in a transgenic mice model. In situ hybridization indicated that connexin43 (Cx43) mRNA was highly expressed either at early tumorogenesis (3 m) characterized by intense proliferation of Leydig cells, or at advanced tumorogenesis (6-7 m) when tumor cells completely invaded the testis. In contrast, Cx43 protein analyzed by Western blotting or classic immunohistochemical analyses was present at the beginning of tumor progression, but was dramatically reduced as tumor advanced. Application of high-resolution deconvolution microscopy to testis sections demonstrates that cells that proliferate exhibited an aberrant cytoplasmic Cx43 localization, in contrast to the expected plasma membrane Cx43 localization in normal Leydig cells. Dual immunofluorescence labeling with specific markers of cellular compartments shows that cytoplasmic Cx43 signal was mainly sequestered within early endosomes. Altogether, this study provides the first evidence that impaired Cx43 trafficking in endosomes is an early event associated with uncontrolled cell proliferation that could serve as a neoplastic marker.