Intracellular transport, cell morphogenesis, motility, and division require the precise control of the microtubule (MT) cytoskeleton, which varies in shape and dynamic. Drosophila oogenesis requires the formation of cortical growing MTs that produces MTs twisters, sustaining cytoplasmic advection and polarization. Although Kinesin-1 MT motor is a central player regulating these processes, its exact function in the formation of these MTs is elusive. Here, we show that Ensconsin/MAP7, a Kinesin-1 activator, is transported by Dynein together with MTs and maintained at the oocyte cortex by Ninein to regulate spatial activation of Kinesin-1 in the oocyte. Perturbation of this process leads to a severe reduction of ncMTOCs (non-centrosomal MicroTubule Organizing Centers) targeting to the cell cortex, and reduced MTs anchoring and stabilization. We also show that an active Khc variant, unable to bind Ens/MAP7 and harboring a loss of auto-inhibition, is sufficient to restore ncMTOCs recruitment, MTs twister formation and advection in the oocyte. Our findings reveal a pivotal mechanism by which the targeted localization of activators drives the spatial activation of motors, a process fundamental to microtubule remodeling. ### Competing Interest Statement The authors have declared no competing interest.

The maintenance of a restricted pool of asymmetrically dividing stem cells is essential for tissue homeostasis. This process requires the control of mitotic progression that ensures the accurate chromosome segregation. In addition, this event is coupled to the asymmetric distribution of cell fate determinants in order to prevent stem cell amplification. How this coupling is regulated remains poorly described. Here, using asymmetrically dividing Drosophila neural stem cells (NSCs), we show that Polo kinase activity levels determine timely Cyclin B degradation and mitotic progression independent of the spindle assembly checkpoint (SAC). This event is mediated by the direct phosphorylation of Polo kinase by Aurora A at spindle poles and Aurora B kinases at centromeres. Furthermore, we show that Aurora A-dependent activation of Polo is the major event that promotes NSC polarization and together with the SAC prevents brain tumor growth. Altogether, our results show that an Aurora/Polo kinase module couples NSC mitotic progression and polarization for tissue homeostasis.

Proper mitotic spindle orientation depends on the correct anchorage of astral microtubules to the cortex. It relies on the remodeling of the cell cortex, a process not fully understood. Annexin A2 (Anx2; also known as ANXA2) is a protein known to be involved in cortical domain remodeling. Here, we report that in HeLa cell early mitosis, Anx2 recruits the scaffold protein Ahnak at the cell cortex facing spindle poles, and the distribution of both proteins is controlled by cell adhesion. Depletion of either protein or impaired cortical Ahnak localization result in delayed anaphase onset and unstable spindle anchoring, which leads to altered spindle orientation. We find that Ahnak is present in a complex with dynein-dynactin. Furthermore. Ahnak and Anx2 are required for correct dynein and NuMA (also known as NUMA1) cortical localization and dynamics. We propose that the Ahnak-Anx2 complex influences the cortical organization of the astral microtubule-anchoring complex, and thereby mitotic spindle positioning in human cells. This article has an associated First Person interview with the first author of the paper.

First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Aude Pascal is first author on `Annexin A2 and Ahnak control cortical NuMA-dynein localization and mitotic spindle orientation', published in JCS. Aude is a research assistant in the lab of Re ' gis Giet at University of Rennes, France, who is particularly interested in developmental biology. She has always been struck by the fact that a whole organism displaying multiple functions arises from a single cell. For this reason, she has oriented her research on mitosis and meiosis to study the different steps, components and structures involved in these processes.

Neuroblast division is characterized by asymmetric positioning of the cleavage furrow, resulting in a large difference in size between the future daughter cells. In animal cells, furrow placement and assembly are governed by centralspindlin that accumulates at the equatorial cell cortex of the future cleavage site and at the spindle midzone. In neuroblasts, these two centralspindlin populations are spatially and temporally separated. A leading pool is located at the basal cleavage site and a second pool accumulates at the midzone before traveling to the cleavage site. The cortical centralspindlin population requires peripheral astral micro tubules and the chromosome passenger complex for efficient recruitment. Loss of this pool does not prevent cytokinesis but enhances centralspindlin signaling at the midzone, leading to equatorial furrow repositioning and decreased size asymmetry. These data show that basal furrow positioning in neuroblasts results from a competition between different centralspindlin pools in which the cortical pool is dominant.

Proper assembly of mitotic spindles requires microtubule nucleation not only at the centrosomes but also around chromatin. In this study, we found that the Drosophila tubulin-specific chaperone dTBCE is required for the enrichment of tubulin in the nuclear space after nuclear envelope breakdown and for subsequent promotion of spindle microtubule nucleation. These events depend on the CAP-Gly motif found in dTBCE and are regulated by Ran and lamin proteins. Our data suggest that during early mitosis, dTBCE and nuclear pore proteins become enriched in the nucleus, where they interact with the Ran GTPase to promote dynamic tubulin enrichment. We propose that this novel mechanism enhances microtubule nucleation around chromatin, thereby facilitating mitotic spindle assembly.

Drosophila Ensconsin (also known as MAP7) controls spindle length, centrosome separation in brain neuroblasts (NBs) and asymmetric transport in oocytes. The control of spindle length by Ensconsin is Kinesin-1 independent but centrosome separation and oocyte transport require targeting of Kinesin-1 to microtubules by Ensconsin. However, the molecular mechanism used for this targeting remains unclear. Ensconsin contains a microtubule (MT)-binding domain (MBD) and a Kinesin-binding domain (KBD). Rescue experiments show that only full-length Ensconsin restores the spindle length phenotype. KBD expression rescues ensc centrosome separation defects in NBs, but not the fast oocyte streaming and the localization of Staufen and Gurken. Interestingly, the KBD can stimulate Kinesin-1 targeting to MTs in vivo and in vitro. We propose that a KBD and Kinesin-1 complex is a minimal activation module that increases Kinesin-1 affinity for MTs. Addition of the MBD present in full-length Ensconsin allows this process to occur directly on the MT and triggers higher Kinesin-1 targeting. This dual regulation by Ensconsin is essential for optimal Kinesin-1 targeting to MTs in oocytes, but not in NBs, illustrating the importance of adapting Kinesin-1 recruitment to different biological contexts.

Centrosome separation in Drosophila larval neuroblasts and asymmetric transport of embryonic determinants in oocytes are both microtubule-dependent processes that require Kinesin-1 activation by Ensconsin/microtubule-associated protein 7 (MAP7). However, the molecular mechanism used by Ensconsin to activate Kinesin-1 remains elusive. Ensconsin/ MAP7 contains an N-terminal microtubule-binding domain (MBD) and a C-terminal Kinesin-binding domain (KBD). Using rescue experiments in live flies, we show that KBD expression alone is sufficient to restore the correct localization patterns of Staufen and Gurken proteins in oocytes. Moreover, in neural stem cells, this domain is also sufficient to fully rescue Ensconsin-dependent centrosome separation defects. Therefore, we propose that the KBD/Kinesin-1 motor represents a minimal activation module that is sufficient to promote Kinesin-1 activation in most Drosophila cells. Nonetheless, our data also suggest that in a very large cell with a complex microtubule network, this minimal module is not sufficient for optimal Kinesin-1 function that requires direct targeting of Kinesin-1 to microtubules by Ensconsin.

AbstractCentrosome separation inDrosophilalarval neuroblasts and asymmetric transport of embryonic determinants in oocytes are both microtubule-dependent processes that require Kinesin-1 activation by Ensconsin/microtubule-associated protein 7 (MAP7). However, the molecular mechanism used by Ensconsin to activate Kinesin-1 remains elusive. Ensconsin/ MAP7 contains an N-terminal microtubule-binding domain (MBD) and a C-terminal Kinesin-binding domain (KBD). Using rescue experiments in live flies, we show that KBD expression alone is sufficient to fully rescue Ensconsin-dependent centrosome separation defects, but not the fast oocyte streaming and the localization patterns of Staufen and Gurken proteins. Interestingly, we show here for the first time that KBD binds and stimulates Kinesin-1 binding to Mtsin vivoandin vitro. We propose that the KBD/Kinesin-1 motor represents a minimal activation module that stimulates Kinesin-1 binding to Mts. Addition of the MBD, present in the full length Ensconsin allows this activation to occur directly on the Mt. Our data also suggest that in a very large cell with a complex microtubule network, but not in smaller cells, this dual activation by Ensconsin is essential for optimal Kinesin-1 targeting to the microtubule cytoskeleton.

Centrosome separation in Drosophila larval neuroblasts and asymmetric transport of embryonic determinants in oocytes are both microtubule-dependent processes that require Kinesin-1 activation by Ensconsin/microtubule-associated protein 7 (MAP7). However, the molecular mechanism used by Ensconsin to activate Kinesin-1 remains elusive. Ensconsin/ MAP7 contains an N-terminal microtubule-binding domain (MBD) and a C-terminal Kinesin-binding domain (KBD). Using rescue experiments in live flies, we show that KBD expression alone is sufficient to restore the correct localization patterns of Staufen and Gurken proteins in oocytes. Moreover, in neural stem cells, this domain is also sufficient to fully rescue Ensconsin-dependent centrosome separation defects. Therefore, we propose that the KBD/Kinesin-1 motor represents a minimal activation module that is sufficient to promote Kinesin-1 activation in most Drosophila cells. Nonetheless, our data also suggest that in a very large cell with a complex microtubule network, this minimal module is not sufficient for optimal Kinesin-1 function that requires direct targeting of Kinesin-1 to microtubules by Ensconsin.

Centrosome separation in Drosophila larval neuroblasts and asymmetric transport of embryonic determinants in oocytes are both microtubule-dependent processes that require Kinesin-1 activation by Ensconsin/microtubule-associated protein 7 (MAP7). However, the molecular mechanism used by Ensconsin to activate Kinesin-1 remains elusive. Ensconsin/ MAP7 contains an N-terminal microtubule-binding domain (MBD) and a C-terminal Kinesin-binding domain (KBD). Using rescue experiments in live flies, we show that KBD expression alone is sufficient to fully rescue Ensconsin-dependent centrosome separation defects, but not the fast oocyte streaming and the localization patterns of Staufen and Gurken proteins. Interestingly, we show here for the first time that KBD binds and stimulates Kinesin-1 binding to Mts in vivo and in vitro . We propose that the KBD/Kinesin-1 motor represents a minimal activation module that stimulates Kinesin-1 binding to Mts. Addition of the MBD, present in the full length Ensconsin allows this activation to occur directly on the Mt. Our data also suggest that in a very large cell with a complex microtubule network, but not in smaller cells, this dual activation by Ensconsin is essential for optimal Kinesin-1 targeting to the microtubule cytoskeleton.

Tissue homeostasis requires accurate control of cell proliferation, differentiation and chromosome segregation. Drosophila sas-4 and aurA mutants present brain tumours with extra neuroblasts (NBs), defective mitotic spindle assembly and delayed mitosis due to activation of the spindle assembly checkpoint (SAC). Here we inactivate the SAC in aurA and sas-4 mutants to determine whether the generation of aneuploidy compromises NB proliferation. Inactivation of the SAC in the sas-4 mutant impairs NB proliferation and disrupts euploidy. By contrast, disrupting the SAC in the aurA mutant does not prevent NB amplification, tumour formation or chromosome segregation. The monitoring of Mad2 and cyclin B dynamics in live aurA NBs reveals that SAC satisfaction is not coupled to cyclin B degradation. Thus, the NBs of aurA mutants present delayed mitosis, with accurate chromosome segregation occurring in a SAC-independent manner. We report here the existence of an Aurora A-dependent mechanism promoting efficient, timed cyclin B degradation.

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.

MAP kinases of the ERK family play important roles in oocyte maturation, fertilization, and early embryo development. The role of the signaling pathway involving ERK5 MAP kinase during meiotic and mitotic M-phase of the cell cycle is not well known. Here, we studied the localization of the phosphorylated, and thus potentially activated, form of ERK5 in mouse maturing oocytes and mitotically dividing early embryos. We show that phosphorylation/dephosphorylation, i.e. likely activation/inactivation of ERK5, correlates with M-phase progression. Phosphorylated form of ERK5 accumulates in division spindle of both meiotic and mitotic cells, and precisely co-localizes with spindle microtubules at metaphase. This localization changes drastically in the anaphase, when phospho-ERK5 completely disappears from microtubules and transits to the cytoplasmic granular, vesicle-like structures. In telophase oocytes it becomes incorporated into the midbody. Dynamic changes in the localization of phospho-ERK5 suggests that it may play an important role both in meiotic and mitotic division.

Background CDK11p58 is a mitotic protein kinase, which has been shown to be required for different mitotic events such as centrosome maturation, chromatid cohesion and cytokinesis. Methodology/Principal Findings In addition to these previously described roles, our study shows that CDK11p58 inhibition induces a failure in the centriole duplication process in different human cell lines. We propose that this effect is mediated by the defective centrosomal recruitment of proteins at the onset of mitosis. Indeed, Plk4 protein kinase and the centrosomal protein Cep192, which are key components of the centriole duplication machinery, showed reduced levels at centrosomes of mitotic CDK11-depleted cells. CDK11p58, which accumulates only in the vicinity of mitotic centrosomes, directly interacts with the centriole-associated protein kinase Plk4 that regulates centriole number in cells. In addition, we show that centriole from CDK11 defective cells are not able to be over duplicated following Plk4 overexpression. Conclusion/Significance We thus propose that CDK11 is required for centriole duplication by two non-mutually-exclusive mechanisms. On one hand, the observed duplication defect could be caused indirectly by a failure of the centrosome to fully maturate during mitosis. On the other hand, CDK11p58 could also directly regulate key centriole components such as Plk4 during mitosis to trigger essential mitotic centriole modifications, required for centriole duplication during subsequent interphase.

The capacity of oocytes to fully support meiotic maturation develops gradually during oocyte growth. Growing oocytes accumulate proteins and mRNAs required for this process. However, little is known about the identity of these factors. We performed a differential proteomic screen comparing the proteomes of growing stage-IV oocytes, which do not undergo meiotic maturation in response to progesterone, with fully grown stage-VI ones, which do. In 2D gels of stage-VI oocytes, we identified a group of four protein spots as EP45 (estrogen-regulated protein 45 kDa), which belongs to the family of serine protease inhibitors and is also known as Seryp or pNiXa. Western blot analysis after mono- and bi-dimensional electrophoreses confirmed the accumulation of certain forms of this protein in oocytes between stages IV and VI. EP45 mRNA was not detectable in oocytes or ovaries, but was expressed in the liver. A low-mobility isoform of EP45 was detected in liver and blood, whereas two (occasionally three or four) higher-mobility isoforms were found exclusively in oocytes, suggesting that liver-synthesized protein is taken up by oocytes from the blood and rapidly modified. Alone, overexpression of RNA encoding either full-length or N-terminally truncated protein had no effect on meiotic resumption in stage-IV or -VI oocytes. However, in oocytes moderately reacting to low doses of progesterone, it significantly enhanced germinal-vesicle breakdown, showing a novel and unsuspected activity of this protein. Thus, EP45 accumulates in growing oocytes through uptake from the blood and has the capacity to act as an ‘oocyte-maturation enhancer’ (‘Omen’).

Aurora A is a spindle pole-associated protein kinase required for mitotic spindle assembly and chromosome segregation. In this study, we show that Drosophila melanogaster aurora A phosphorylates the dynactin subunit p150(glued) on sites required for its association with the mitotic spindle. Dynactin strongly accumulates on microtubules during prophase but disappears as soon as the nuclear envelope breaks down, suggesting that its spindle localization is tightly regulated. If aurora A's function is compromised, dynactin and dynein become enriched on mitotic spindle microtubules. Phosphorylation sites are localized within the conserved microtubule-binding domain (MBD) of the p150(glued). Although wildtype p150(glued) binds weakly to spindle microtubules, a variant that can no longer be phosphorylated by aurora A remains associated with spindle microtubules and fails to rescue depletion of endogenous p150(glued). Our results suggest that aurora A kinase participates in vivo to the phosphoregulation of the p150(glued) MBD to limit the microtubule binding of the dynein-dynactin complex and thus regulates spindle assembly.

The quality of oocytes depends largely on the capacity to resume meiotic maturation. In Xenopus laevis, only fully grown oocytes react to progesterone stimulation by resumption of meiotic maturation associated with the entry into the meiotic M-phase. Proteins involved in this process are poorly known. To identify novel proteins regulating M-phase entry, we performed a differential proteomic screen. We compared proteomes of fully grown stage VI oocytes characterized as poorly or highly responsive to progesterone treatment. The comparison of 2-D gels allowed us to identify several spots including two specifically present in highly responsive oocytes and two specifically present in poorly responsive ones. By mass spectrometry we identified the two proteins specifically present in highly responsive oocytes as inosine 5′monophosphate cyclohydrolase and YjgF homologues, and the two specifically present in poorly responsive oocytes as elongation factor 2 (EF2) and S-adenosyl-l-homocysteine hydrolase (SAHH). The proteins specifically expressed in highly responsive oocytes may participate in the stimulation of meiotic maturation and M-phase entry, while the proteins specifically present in poorly maturing oocytes may participate in the inhibition of meiotic resumption.