Microtubule-organizing Center Research Articles

Betrayal From the Core: Centriolar and Cytoskeletal Subversion by Infectious Pathogens.

Microbes and parasites have evolved several means to evade and usurp the host cellular machinery to mediate pathogenesis. Being the major microtubule-organizing center (MTOC) of the cell, the centrosome is targeted by multiple viral and nonviral pathogens to mediate their assembly and trafficking within the host cell. This review examines the consequence of such targeting to the centrosome and associated cytoskeletal machinery. We have also amassed a substantial body of evidence of viruses utilizing the cilia within airway epithelium to mediate infection and the hijacking of host cytoskeletal machinery for efficient entry, replication, and egress. While infections have been demonstrated to induce structural, functional, and numerical aberrations in centrosomes, and induce ciliary dysfunction, current literature increasingly supports the notion of a pro-viral role for these organelles. Although less explored, the impact of bacterial and parasitic pathogens on these structures has also been addressed very briefly. Mechanistically, the molecular pathways responsible for these effects remain largely uncharacterized in many instances. Future research focusing on the centriolar triad comprising the centrosome, cilia, and centriolar satellites will undoubtedly provide vital insights into the tactics employed by infectious agents to subvert the host centriole and cytoskeleton-based machinery.

Triptolide exposure triggers ovarian inflammation by activating cGAS-STING pathway and decrease oocyte quality in mouse.

Triptolide (TPL), a prominent bioactive constituent derived from the Chinese herb Tripterygium wilfordii, exhibits diverse pharmacological effects such as anti-tumor and anti-immune properties. Despite its extensive clinical application for the treatment of arthritis and immune disorders, TPL has been associated with multiorgan toxicity, including adverse effects on the female reproductive system. However, the precise mechanisms underlying TPL-induced ovarian damage remain poorly understood. In this study, employing a mouse toxicological model, exposure to TPL was observed to result in decreased ovarian coefficient and fertility. Subsequent research demonstrated TPL exposure affected mitochondrial function, increased mitochondrial outer membrane permeability, resulted in mtDNA releasing into the cytoplasm. These events subsequently activated cGAS-STING pathway, leading to ovarian inflammation. Furthermore, TPL exposure has been found to disrupt the meiotic maturation of oocytes, which is mechanistically associated with suboptimal morphology of spindle and microtubule organizing centers (MTOCs). This association has been further confirmed through the use of reduced representation bisulfite sequencing (RRBS). In conclusion, our study demonstrates that TPL exposure can hinder follicular development, resulting in ovarian inflammation and reduced oocyte quality.

CLASP1/2 REGULATE IMMUNE SYNAPSE MATURATION IN NATURAL KILLER CELLS.

The Golgi apparatus (GA) functions as a microtubule-organizing center (MTOC) in NK cells. During the recognition of tumoral cells by NK cells, CLASP1/2-mediated stabilization of GA-derived microtubules (GDMTs) facilitates vesicular LFA-1 (LFA-1 v ) trafficking toward the interaction surface, thereby promoting the immune synapse (IS) maturation.

Peri-centrosomal localization of small interfering RNAs in C. elegans.

The centrosome is the microtubule-organizing center and a crucial part of cell division. Centrosomal RNAs (cnRNAs) have been reported to enable precise spatiotemporal control of gene expression during cell division in many species. Whether and how cnRNAs exist in C. elegans are unclear. Here, using the nuclear RNAi Argonaute protein NRDE-3 as a reporter, we observed potential peri-centrosome localized small interfering (si)RNAs in C. elegans. NRDE-3 was previously shown to associate with pre-mRNAs and pre-rRNAs via a process involving the presence of complementary siRNAs. We generated a GFP-NRDE-3 knock-in transgene through CRISPR/Cas9 technology and observed that NRDE-3 formed peri-centrosomal foci neighboring the tubulin protein TBB-2, other centriole proteins and pericentriolar material (PCM) components in C. elegans embryos. The peri-centrosomal accumulation of NRDE-3 depends on RNA-dependent RNA polymerase (RdRP)-synthesized 22G siRNAs and the PAZ domain of NRDE-3, which is essential for siRNA binding. Mutation of eri-1, ergo-1, or drh-3 significantly increased the percentage of pericentrosome-enriched NRDE-3. At the metaphase of the cell cycle, NRDE-3 was enriched in both the peri-centrosomal region and the spindle. Moreover, the integrity of centriole proteins and pericentriolar material (PCM) components is also required for the peri-centrosomal accumulation of NRDE-3. Therefore, we concluded that siRNAs could accumulate in the pericentrosomal region in C. elegans and suggested that the peri-centrosomal region may also be a platform for RNAi-mediated gene regulation.

Insufficient MIRO1 contributes to declined oocyte quality during reproductive aging.

Mitochondrial Rho-GTPase 1 (MIRO1) is an outer mitochondrial membrane protein which regulates mitochondrial transport and mitophagy in mitosis. In present study, we reported the crucial roles of MIRO1 in mammalian oocyte meiosis and its potential relationship with aging. We found that MIRO1 expressed in mouse and porcine oocytes, and its expression decreased in aged mice. MIRO1 deficiency caused the failure of meiotic resumption and polar body extrusion in both mouse and porcine oocytes, which could be rescued by exogenous MIRO1 supplementation. Mass spectrometry data indicated that MIRO1 associated with several cytoskeleton and cell cycle-related proteins, and MIRO1 regulated motor protein Dynein for microtubule-organizing centers (MTOCs) dynamics at germinal vesicle (GV) stage, which determined meiotic resumption. Furthermore, we found that MIRO1 regulated Aurora A and kinesin family member 11 (KIF11) for meiotic spindle assembly in oocytes. Besides, MIRO1 associated with several mitochondria-related proteins dynamic-related protein 1 (DRP1), Parkin and lysosomal-associated membrane protein 2 (LAMP2) for mitochondrial dynamics and mitophagy during oocyte meiosis. Taken together, our results suggested that MIRO1 played pivotal roles in meiotic resumption, spindle assembly and mitochondrial function in mouse and porcine oocytes, and its insufficiency might contribute to the oocyte maturation defects during aging.

The Golgi complex governs natural killer cell lytic granule positioning to promote directionality in cytotoxicity.

Cytotoxic immune cells mediate precise attacks against diseased cells to maintain organismal health. Their operational unit of killing and host defense is lytic granules (LGs), which are specialized lysosomal-related organelles. Precision in cytotoxicity is achieved by converging the many LGs to the microtubule-organizing center (MTOC) and polarizing these to the diseased cell for secretion. We identify unappreciated intimate relationships between the Golgi, MTOC, and LGs after cytotoxic cell activation, as well as the trans-Golgin protein GCC2 on the LG surface. GCC2 serves to tether LGs to the Golgi following convergence, and both GCC2 and the Golgi are required for the persistence of convergence. GCC2 allows LGs to utilize the Golgi as a docking station preventing LG dispersion and innocent bystander killing in complex three-dimensional environments. We also identify GCC2 variants causing human natural killer cell deficiency, further emphasizing the importance of LG convergence and Golgi linkage in precision targeting for human immunity.

Visualizing Cartwheel Disassembly Process During Mitosis in Fixed and Live Cells by Fluorescence Microscope.

Centrosome is an evolutionarily conserved organelle that comprises two barrel-shaped centrioles surrounded by pericentriolar material (PCM). It functions as the major microtubule-organizing center (MTOC) to regulate cell polarity, motility, intracellular material transport during interphase, and bipolar spindle assembly during mitosis. Cartwheel assembly is considered the first step in the initiation of procentriole biogenesis at early S phase. In human cells, cartwheel is a transient scaffolding structure that is disassembled during procentriole maturation at mitotic exit. This chapter describes methods for synchronizing mammalian cells into mitosis by small molecule inhibitor treatment and visualizing cartwheel disassembly process during mitosis in fixed and live cells by fluorescence microscope.

MLL/WDR5 complex recruits centriolar satellite protein Cep72 to regulate microtubule nucleation and spindle formation.

Dysfunction of the centrosome, the major microtubule-organizing center of the cell, is implicated in microcephaly. Haploinsufficiency of mixed-lineage leukemia (MLL/KMT2A) protein causes Wiedemann-Steiner syndrome (WSS), a neurodevelopmental disorder associated with microcephaly. However, whether MLL has a function at the centrosome is not clear. Here, we show that loss of the MLL/WDR5 complex affects microtubule nucleation and regrowth. MLL/WDR5 localize to the pericentriolar material and interact with centriolar satellite protein Cep72 and γ-tubulin ring complex proteins (γ-TuRCs). MLL/WDR5 promote the localization of γ-TuRCs and structural proteins like AKAP9 to the centrosome during interphase and mitosis, a phenotype also observed in cells derived from patients with WSS. During mitosis, loss of MLL, WDR5, and Cep72 affects spindle formation and leads to misaligned chromosomes. Last, we show that MLL and WDR5 recruit Cep72 to the centrosome. Our studies provide insight into an undiscovered role of MLL at the centrosome and elucidate how centriolar satellite proteins like Cep72 can be recruited to the centrosome.

Structure of the microtubule anchoring factor NEDD1 bound to the γ-tubulin ring complex.

The γ-tubulin ring complex (γ-TuRC) is an essential multiprotein assembly, in which γ-tubulin, GCP2-6, actin, MZT1 and MZT2 form an asymmetric cone-shaped structure that provides a template for microtubule nucleation. The γ-TuRC is recruited to microtubule organizing centers (MTOCs), such as centrosomes and pre-existing mitotic spindle microtubules, via the evolutionarily-conserved attachment factor NEDD1. NEDD1 contains an N-terminal WD40 domain that binds to microtubules, and a C-terminal domain that associates with the γ-TuRC. However, the structural basis of the NEDD1-γ-TuRC interaction is not known. Here, we report cryo-electron microscopy (cryo-EM) structures of NEDD1 bound to the human γ-TuRC in the absence or presence of the activating factor CDK5RAP2, which interacts with GCP2 to induce conformational changes in the γ-TuRC and promote its microtubule nucleating function. We found that the C-terminus of NEDD1 forms a tetrameric α-helical assembly that contacts the lumen of the γ-TuRC cone, is anchored to GCP4, 5 and 6 via protein modules consisting of MZT1 & GCP3 subcomplexes, and orients its microtubule-binding WD40 domains away from the complex. We biochemically tested our structural models by identifying NEDD1 mutants unable to pull-down γ-tubulin from cultured cells. The structure of the γ-TuRC simultaneously bound to NEDD1 and CDK5RAP2 reveals that both factors can associate with the "open" conformation of the complex. Our results show that NEDD1 does not induce conformational changes in the γ-TuRC, but suggest that anchoring of γ-TuRC-capped microtubules by NEDD1 would be structurally compatible with the significant conformational changes experienced by the γ-TuRC during microtubule nucleation.

Formin-like 1β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes

We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.

Formin-like 1β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes.

We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.

Versatile gamma-tubulin complexes contribute to the dynamic organization of MTOCs during Drosophila spermatogenesis

The initiation of microtubule formation is facilitated by γ-tubulin and γ-Tubulin Ring Complex (γ-TuRC) in various microtubule-organizing centers (MTOCs). While the heterogeneity of tissue-specific MTOCs and γ-TuRC in Drosophila testis has been described, their molecular composition and physiological significance are poorly understood. We investigated the testis-specific distribution and biochemical interaction of the canonical γ-TuRC proteins Grip163 and Grip84. We found that while Grip163 is present on the centrosome and basal body, Grip84 localizes to the centrosome and Golgi in spermatocytes and colocalizes with the testis-specific γ-Tubulin complexes (t-γ-TuC) at the basal body, apical nuclear tip, and near the elongated mitochondria after meiosis. We also showed the apical nuclear tip localization of some γ-TuRC interacting partners and proved their binding to t-γ-TuC proteins. These results highlight and prove the importance of the different γ-TuRCs in organizing the diverse MTOCs present during the extensive rearrangement of cell organelles during the spermatogenesis of Drosophila.

PAK4 is Required for Meiotic Resumption, Spindle Assembly, and Cortical Migration in Mouse Oocytes During Meiotic Maturation.

Oocyte meiotic errors can cause infertility, miscarriage, and birth defects. Here the role and the underlying mechanism of p21 activated kinase 4 (PAK4) in mouse oocyte meiosis is evaluated. It is found that PAK4 expression and its phosphorylation are detected in high level at germinal vesicle (GV) stage, and gradually decreased after meiotic resumption in oocytes. PAK4 has direct physical interaction with both mitogen-activated protein kinases 1/2 (MEK1/2) and Paxillin, they are colocalized on the spindle structure during metaphases I and II. Phospho-PAK4 is distributed beneath the cytoplasmic membrane and on the chromosomes, and colocalized with the microtubule organizing center (MTOC) proteins, Pericentrin and γ-tubulin, as well as phosphor-MEK1/2 and phosphor-Paxillin on spindle poles. PAK4 inhibition by chemical inhibitor LCH-7749944, specific Pak4 morpholino oligo or the dominant negative mutant Pak4K350, 351M influence the meiotic resumption, spindle assembly and its cortical migration, and associated with the downregulation in the dephosphorylation of cyclin dependent kinase 1 (CDK1) and the levels of Cyclin B1, MEK1/2, Paxillin, g-tubulin, acetylated a-tubulin, Arp3, and Cofilin phosphorylation in oocytes. In sum, PAK4 functions to sustain the rational levels of Cyclin B1, MEK1/2, Paxillin, y-tubulin, acetylated a-tubulin, Arp3, and phosphor-Cofilin in mouse oocytes, thereby promotes the meiotic resumption, spindle assembly, and migration during meiotic maturation.

A multistage Plasmodium CRL4WIG1 ubiquitin ligase is critical for the formation of functional microtubule organization centers in microgametocytes.

Malaria is a mosquito-borne infectious disease caused by unicellular eukaryotic parasites of the Plasmodium genus. Protein ubiquitination by E3 ligases is a critical post-translational modification required for various cellular processes during the lifecycle of Plasmodium parasites. However, little is known about the repertoire and function of these enzymes in Plasmodium. Here, we show that Plasmodium expresses a conserved cullin RING E3 ligase (CRL) complex that is functionally related to CRL4 in other eukaryotes. In P. falciparum asexual blood stages, a cullin-4 scaffold interacts with the RING protein RBX1, the adaptor protein DDB1, and a set of putative receptor proteins that may determine substrate specificity for ubiquitination. These receptor proteins contain WD40-repeat domains and include WD-repeat protein important for gametogenesis 1 (WIG1). This CRL4-related complex is also expressed in P. berghei gametocytes, with WIG1 being the only putative receptor detected in both the schizont and gametocyte stages. WIG1 disruption leads to a complete block in microgamete formation. Proteomic analyses indicate that WIG1 disruption alters proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. Further analysis by ultrastructure expansion microscopy (U-ExM) indicates that WIG1-dependent depletion of ciliary proteins is associated with impaired the formation of the microtubule organization centers that coordinate mitosis with axoneme formation and altered DNA replication during microgametogenesis. This work identifies a CRL4-related ubiquitin ligase in Plasmodium that is critical for the formation of microgametes by regulating proteostasis of ciliary and DNA replication proteins.IMPORTANCEPlasmodium parasites undergo fascinating lifecycles with multiple developmental steps, converting into morphologically distinct forms in both their mammalian and mosquito hosts. Protein ubiquitination by ubiquitin ligases emerges as an important post-translational modification required to control multiple developmental stages in Plasmodium. Here, we identify a cullin RING E3 ubiquitin ligase (CRL) complex expressed in the replicating asexual blood stages and in the gametocyte stages that mediate transmission to the mosquito. WIG1, a putative substrate recognition protein of this ligase complex, is essential for the maturation of microgametocytes into microgametes upon ingestion by a mosquito. More specifically, WIG1 is required for proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. This requirement is linked to DNA replication and microtubule organization center formation, both critical to the development of flagellated microgametes.

TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly.

Tightly controlled duplication of centrosomes, the major microtubule-organizing centers of animal cells, ensures bipolarity of the mitotic spindle and accurate chromosome segregation. The RBCC (RING-B-box-coiled coil) ubiquitin ligase TRIM37, whose loss is associated with elevated chromosome missegregation and the tumor-prone developmental human disorder Mulibrey nanism, prevents the formation of ectopic spindle poles that assemble around structured condensates containing the centrosomal protein centrobin. Here, we show that TRIM37's TRAF domain, unique in the extended TRIM family, engages peptide motifs in centrobin to suppress condensate formation. TRIM proteins form anti-parallel coiled-coil dimers with RING-B-box domains on each end. Oligomerization due to RING-RING interactions and conformational regulation by B-box-2-B-box-2 interfaces are critical for TRIM37 to suppress centrobin condensate formation. These results indicate that, analogous to anti-viral TRIM ligases, TRIM37 activation is linked to the detection of oligomerized substrates. Thus, TRIM37 couples peptide motif recognition and substrate-dependent oligomerization to effect ubiquitination-mediated clearance of ectopic centrosomal protein assemblies.

B cell mechanosensing regulates ER remodeling at the immune synapse.

Engagement of the B-cell receptor with immobilized antigens triggers the formation of an immune synapse (IS), a complex cellular platform where B-cells recruit signaling molecules and reposition lysosomes to promote antigen uptake and processing. Calcium efflux from the endoplasmic reticulum (ER) released upon BCR stimulation is necessary to promote B-cell survival and differentiation. Whether the spatial organization of the ER within the B-cell synapse can tune IS function and B-cell activation remains unaddressed. Here, we characterized ER structure and interaction with the microtubule network during BCR activation and evaluated how mechanical cues arising from antigen presenting surfaces affect this process. B-cells were cultured on surfaces of varying stiffness coated with BCR ligands, fixed, and stained for the ER and microtubule network. Imaging analysis was used to assess the distribution of the ER and microtubules at the IS. Upon BCR activation, the ER is redistributed towards the IS independently of peripheral microtubules and accumulates around the microtubule-organization center. Furthermore, this remodeling is also dependent on substrate stiffness, where greater stiffness triggers enhanced redistribution of the ER. Our results highlight how spatial reorganization of the ER is coupled to the context of antigen recognition and could tune B-cell responses. Additionally, we provide novel evidence that the structural maturation of the ER in plasma cells is initiated during early activation of B-cells.

Tail-anchored membrane protein SLMAP3 is essential for targeting centrosomal proteins to the nuclear envelope in skeletal myogenesis.

The positioning and communication between the nucleus and centrosomes are essential in cell division, differentiation and tissue formation. During skeletal myogenesis, the nuclei become evenly spaced with the switch of the microtubule-organizing centre (MTOC) from the centrosome to the nuclear envelope (NE). We report that the tail-anchored sarcolemmal membrane associated protein 3 (SLMAP3), a component of the MTOC and NE, is crucial for myogenesis because its deletion in mice leads to a reduction in the NE-MTOC formation, mislocalization of the nuclei, dysregulation of the myogenic programme and abnormal embryonic myofibres. SLMAP3-/- myoblasts also displayed a similar disorganized distribution of nuclei with an aberrant NE-MTOC and defective myofibre formation and differentiation programming. We identified novel interactors of SLMAP3, including pericentrin, PCM1 (pericentriolar material 1), AKAP9 (A-kinase anchoring protein 9), kinesin-1 members Kif5B (kinesin family member 5B), KCL1 (kinesin light chain 1), KLC2 (kinesin light chain 2) and nuclear lamins, and observed that the distribution of centrosomal proteins at the NE together with Nesprin-1 was significantly altered by the loss of SLMAP3 in differentiating myoblasts. SLMAP3 is believed to negatively regulate Hippo signalling, but its loss was without impact on this pathway in developing muscle. These results reveal that SLMAP3 is essential for skeletal myogenesis through unique mechanisms involving the positioning of nuclei, NE-MTOC dynamics and gene programming.

Human NLRP3 inflammasome activation leads to formation of condensate at the microtubule organizing center.

The NLRP3 inflammasome is a multi-protein molecular machine that mediates inflammatory responses in innate immunity. Its dysregulation has been linked to a large number of human diseases. Using cryogenic fluorescence-guided focused-ion-beam (cryo-FIB) milling and electron cryo-tomography (cryo-ET), we obtained 3-D images of the NLRP3 inflammasome in situ at various stages of its activation at macromolecular resolution. The cryo-tomograms unexpectedly reveal dense condensates of the human macrophage NLRP3 inflammasome that form within and around the microtubule organizing center (MTOC). We also find that following activation, the trans-Golgi network disperses and 50-nm NLRP3-associated vesicles appear which likely ferry NLRP3 to the MTOC. At later time points after activation, the electron-dense condensates progressively solidify and the cells undergo pyroptosis with widespread damaged mitochondria and autophagasomal structures.

Plasmodium NEK1 coordinates MTOC organisation and kinetochore attachment during rapid mitosis in male gamete formation.

Mitosis is an important process in the cell cycle required for cells to divide. Never in mitosis (NIMA)-like kinases (NEKs) are regulators of mitotic functions in diverse organisms. Plasmodium spp., the causative agent of malaria is a divergent unicellular haploid eukaryote with some unusual features in terms of its mitotic and nuclear division cycle that presumably facilitate proliferation in varied environments. For example, during the sexual stage of male gametogenesis that occurs within the mosquito host, an atypical rapid closed endomitosis is observed. Three rounds of genome replication from 1N to 8N and successive cycles of multiple spindle formation and chromosome segregation occur within 8 min followed by karyokinesis to generate haploid gametes. Our previous Plasmodium berghei kinome screen identified 4 Nek genes, of which 2, NEK2 and NEK4, are required for meiosis. NEK1 is likely to be essential for mitosis in asexual blood stage schizogony in the vertebrate host, but its function during male gametogenesis is unknown. Here, we study NEK1 location and function, using live cell imaging, ultrastructure expansion microscopy (U-ExM), and electron microscopy, together with conditional gene knockdown and proteomic approaches. We report spatiotemporal NEK1 location in real-time, coordinated with microtubule organising centre (MTOC) dynamics during the unusual mitoses at various stages of the Plasmodium spp. life cycle. Knockdown studies reveal NEK1 to be an essential component of the MTOC in male cell differentiation, associated with rapid mitosis, spindle formation, and kinetochore attachment. These data suggest that P. berghei NEK1 kinase is an important component of MTOC organisation and essential regulator of chromosome segregation during male gamete formation.

Ninein domains required for its localization, association with partners dynein and ensconsin, and microtubule organization.

Ninein (Nin) is a microtubule (MT) anchor at the subdistal appendages of mother centrioles and the pericentriolar material (PCM) of centrosomes that also functions to organize MTs at noncentrosomal MT-organizing centers (ncMTOCs). In humans, the NIN gene is mutated in Seckel syndrome, an inherited developmental disorder. Here, we dissect the protein domains involved in Nin's localization and interactions with dynein and ensconsin (ens/MAP7) and show that the association with ens cooperatively regulates MT assembly in Drosophila fat body cells. We define domains of Nin responsible for its localization to the ncMTOC on the fat body cell nuclear surface, localization within the nucleus, and association with Dynein light intermediate chain (Dlic) and ens, respectively. We show that Nin's association with ens synergistically regulates MT assembly. Together, these findings reveal novel features of Nin function and its regulation of a ncMTOC.