Microtubule-organizing Center Research Articles

Tunable intracellular transport on converging microtubule morphologies

A common type of cytoskeletal morphology involves multiple microtubules converging with their minus ends at the microtubule organizing center (MTOC). The cargo-motor complex will experience ballistic transport when bound to microtubules or diffusive transport when unbound. This machinery allows for sequestering and subsequent dispersal of dynein-transported cargo. The general principles governing dynamics, efficiency, and tunability of such transport in the MTOC vicinity are not fully understood. To address this, we develop a one-dimensional model that includes advective transport toward an attractor (such as the MTOC) and diffusive transport that allows particles to reach absorbing boundaries (such as cellular membranes). We calculated the mean first passage time (MFPT) for cargo to reach the boundaries as a measure of the effectiveness of sequestering (large MFPT) and diffusive dispersal (low MFPT). We show that the MFPT experiences a dramatic growth, transitioning from a low to high MFPT regime (dispersal to sequestering) over a window of cargo on-/off-rates that is close to in vivo values. Furthermore, increasing either the on-rate (attachment) or off-rate (detachment) can result in optimal dispersal when the attractor is placed asymmetrically. Finally, we also describe a regime of rare events where the MFPT scales exponentially with motor velocity and the escape location becomes exponentially sensitive to the attractor positioning. Our results suggest that structures such as the MTOC allow for the sensitive control of the spatial and temporal features of transport and corresponding function under physiological conditions.

Melatonin mitigates PNMC-induced disruption of spindle assembly and mitochondrial function in mouse Oocytes

3-methyl-4-nitrophenol (PNMC), a degradation product of organophosphorus insecticides and a byproduct of fuel combustion, exerting endocrine-disrupting effects. However, its impact on the meiotic process of oocytes remains unclear. In the present study, we investigated the effects of PNMC on meiotic maturation of mouse oocytes in vitro and related mechanisms. Morphologically, PNMC-exposure affected germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) in mouse oocytes. Proteomic analysis suggested that PNMC-exposure altered oocyte protein expression that are associated with cytoskeleton, mitochondrial function and oxidative stress. Further studies demonstrated that PNMC-exposure disrupted spindle assembly and chromosome alignment, caused sustained activation of spindle assembly checkpoint (SAC), and arrested meiosis in oocytes. Specifically, PNMC-exposure interfered with the function of microtubule organizing centers (MTOCs) by significantly reducing phosphorylated mitogen activated protein kinase (p-MAPK) expression and disrupting the localization of Pericentrin and p-Aurora A, leading to spindle assembly failure. Besides, PNMC-exposure also increased α-tubulin acetylation, decreased microtubule stability. Moreover, PNMC-exposure impaired mitochondrial function, evidenced by abnormal mitochondrial distribution, decreased mitochondrial membrane potential and ATP levels, release of Cytochrome C into the cytoplasm, and elevated ROS levels. As a result, exposure to PNMC caused DNA damage and early apoptosis in oocytes. Fortunately, melatonin was able to promote oocyte maturation by removing the excessive ROS and enhancing mitochondrial function. These results highlight the adverse effects of PNMC on meiotic maturation, and underscore the protective role of melatonin against PNMC-induced damage.

Novel autophagy inducers by accelerating lysosomal clustering against Parkinson's disease.

The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including α-synuclein (αSyn) associated with the pathogenesis of Parkinson's disease (PD). However, the activation of this pathway is mainly by targeting lysosomal enzymic activity. Here, we focused on the autophagosome-lysosome fusion process around the microtubule-organizing center (MTOC) regulated by lysosomal positioning. Through high-throughput chemical screening, we identified 6 out of 1200 clinically approved drugs enabling the lysosomes to accumulate around the MTOC with autophagy flux enhancement. We further demonstrated that these compounds induce the lysosomal clustering through a JIP4-TRPML1-dependent mechanism. Among them, the lysosomal-clustering compound albendazole promoted the autophagy-dependent degradation of Triton-X-insoluble, proteasome inhibitor-induced aggregates. In a cellular PD model, albendazole boosted insoluble αSyn degradation. Our results revealed that lysosomal clustering can facilitate the breakdown of protein aggregates, suggesting that lysosome-clustering compounds may offer a promising therapeutic strategy against neurodegenerative diseases characterized by the presence of aggregate-prone proteins.

O-008 Sperm-derived centrioles play a key role in ensuring correct chromosome segregation in the first mitotic division of mammalian zygotes

Abstract Study question What is the role of the centrosomes during mitotic division in the mammalian zygote? Does the atypical structure of sperm-derived centrioles affect their function? Summary answer Correct centrosome positioning guarantees spindle stability and correct chromosome segregation. Centrosome malposition or inactivity predisposes to chromosome segregation errors and failure of cytokinesis. What is known already The centrosomes of somatic cells comprise two centrioles surrounded by pericentriolar material, and are major microtubule-organizing centres with an essential role in spindle assembly and chromosome segregation. Mammalian oocytes lack centrosomes and, although two centrioles are introduced by the spermatozoon at fertilization, they are structurally different to the canonical centrioles found in somatic cells. We recently demonstrated that centrosomes play a minor role during zygotic spindle assembly, when they are often mal-positioned (not at the spindle poles). However, the causes and consequences of abnormal centrosome positioning in zygotes are not known. Study design, size, duration We combined systematic immunofluorescence of bovine zygotes, fixed at different stages, with real-time imaging of live zygotes to investigate the implications of centrosome mal-positioning during zygotic mitosis on the fidelity of chromosome segregation and cytokinesis. To study the functionality of the sperm-derived centrioles alone, we inhibited the formation of daughter centrioles using the PLK4 inhibitor (PLK4i), Centrinone. Bovine zygotes were used because the structure of bovine and human sperm-derived centrioles are very similar. Participants/materials, setting, methods Bovine oocytes were retrieved from slaughterhouse ovaries and matured in vitro. Matured oocytes were fertilized in vitro with semen from a bull of proven fertility and, either fixed at 27-29h post-fertilization, or used for time-lapse light-sheet imaging after injection of mRNA for microtubule and chromatin reporters. Fixed bovine zygotes were immunostained for chromatin, alpha-tubulin, centriolar and pericentriolar components, and imaged with a confocal microscope. Microtubule nucleation ability was evaluated using a microtubule regrowth assay. Main results and the role of chance Live imaging showed that in 52% of bovine zygotes (24/46) the centrosomes engaged with the spindle but were malposition, while in 26% (12/46) the centrosomes were not visible (inactive). All zygotes with normally positioned centrosomes showed correct chromosome segregation. Centrosome malposition and inactivity induced chromosome segregation errors in 44% and 82% of the zygotes respectively. PLK4i treatment successfully inhibited centriole duplication, since the volume of core-centriole protein (POC5) in treated zygotes was half that in non-treated zygotes. When centriole duplication was prevented, the sperm-derived centrioles showed a similar ability to recruit pericentrosomal material (NEDD1) and nucleate microtubules. However, live imaging showed that 82% (14/17) of PLK4i treated zygotes the spindle, although bipolar, was unusually elongated and chromosomes either failed to align correctly on the metaphase plate or showed anaphase lag. Moreover, in treated zygotes where both centrioles were positioned at the same spindle pole, the spindle failed to anchor correctly to the cell membrane and resulted in rotational behavior of the zygote and an increase of cytokinesis failure from up to 47% (8/17). Therefore we conclude that centrosomes are likely to play a key role in correct chromosome segregation in mammalian zygotes. Limitations, reasons for caution For ethical reasons, we could not manipulate and study human zygotes. Instead, we chose an animal model with a rate of embryonic aneuploidy similar to man. It is, however, possible that zygotic centrosome function differs between these two species. Wider implications of the findings A better understanding of the key regulators of centrosome function might lead to the development of interventions to reduce the risk of malfunction, thereby reducing the incidence of embryonic aneuploidy. Moreover, our study unveiled an mechanism by which impaired PLK4 function induces aneuploidy in mammalian embryos. Trial registration number not applicable

Fbxo28 is essential for spindle migration and morphology during mouse oocyte meiosis I

Spindle migration and assembly regulates asymmetric oocyte division, which is essential for fertility. Fbxo28, as a member of SCF (Skp1-Cul1-F-box) ubiquitin E3 ligases complex, is specifically expressed in oocytes. However, little is known about the functions of Fbxo28 in spindle assembly and migration during oocyte meiosis I. In present study, microinjection with morpholino oligonucleotides and exogenous mRNA for knockdown and rescue experiments, and immunofluorescence staining, western blot, timelapse confocal microscopy and chromosome spreading were utilized to explore the roles of Fbxo28 in asymmetric division during meiotic maturation. Our data suggested that Fbxo28 mainly localized at chromosomes and acentriolar microtubule-organizing centers (aMTOCs). Depletion of Fbxo28 did not affect polar body extrusion but caused defects in spindle morphology and migration, indicative of the failure of asymmetric division. Moreover, absence of Fbxo28 disrupted both cortical and cytoplasmic actin assembly and decreased the expression of ARPC2 and ARP3. These defects could be rescued by exogenous Fbxo28-myc mRNA supplement. Collectively, this study demonstrated that Fbxo28 affects spindle morphology and actin-based spindle migration during mouse oocyte meiotic maturation.

A distinct isoform of Msp300/Nesprin organizes the perinuclear microtubule organizing center in adipose cells.

In many cell types, disparate non-centrosomal microtubule-organizing centers (ncMTOCs) replace functional centrosomes and serve the unique needs of the cell types in which they are formed. In Drosophila fat body cells (adipocytes), an ncMTOC is organized on the nuclear surface. This perinuclear ncMTOC is anchored by Msp300, encoded by one of two Nesprin-encoding genes in Drosophila. Msp300 and the spectraplakin short stop (shot) are co-dependent for localization to the nuclear envelope to generate the ncMTOC where they recruit the microtubule minus-end stabilizer Patronin (CAMSAP). The fat body perinuclear ncMTOC requires Patronin, Ninein, and Msps (ortholog of ch-TOG), but does not require γ-tubulin for MT assembly. The Msp300 gene is complex, encoding at least eleven isoforms. Here we show that two Msp300 isoforms, Msp300-PE and -PG, are required and only one, Msp300-PE, appears sufficient for generation of the ncMTOC. Loss of Msp300-PE,-PG retains the presence of the other isoforms at the nuclear surface, indicating that they are not sufficient to generate the ncMTOC. Loss of Msp300-PE,-PG results in severe loss of localization of shot and Patronin, and disruption of the MT array. This results in nuclear mispositioning and loss of endosomal trafficking. Msp300-PE has an unusual domain structure including a lack of a KASH domain and very few spectrin repeats and appears therefore to have a highly derived function suited to generating an ncMTOC on the nuclear surface.

Spermatocytes have the capacity to segregate chromosomes despite centriole duplication failure

Centrosomes are the canonical microtubule organizing centers (MTOCs) of most mammalian cells, including spermatocytes. Centrosomes comprise a centriole pair within a structurally ordered and dynamic pericentriolar matrix (PCM). Unlike in mitosis, where centrioles duplicate once per cycle, centrioles undergo two rounds of duplication during spermatogenesis. The first duplication is during early meiotic prophase I, and the second is during interkinesis. Using mouse mutants and chemical inhibition, we have blocked centriole duplication during spermatogenesis and determined that non-centrosomal MTOCs (ncMTOCs) can mediate chromosome segregation. This mechanism is different from the acentriolar MTOCs that form bipolar spindles in oocytes, which require PCM components, including gamma-tubulin and CEP192. From an in-depth analysis, we identified six microtubule-associated proteins, TPX2, KIF11, NuMA, and CAMSAP1-3, that localized to the non-centrosomal MTOC. These factors contribute to a mechanism that ensures bipolar MTOC formation and chromosome segregation during spermatogenesis when centriole duplication fails. However, despite the successful completion of meiosis and round spermatid formation, centriole inheritance and PLK4 function are required for normal spermiogenesis and flagella assembly, which are critical to ensure fertility.

INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death

Mutations in the human INF2 gene cause autosomal dominant focal segmental glomerulosclerosis (FSGS)—a condition characterized by podocyte loss, scarring, and subsequent kidney degeneration. To understand INF2-linked pathogenicity, we examined the effect of pathogenic INF2 on renal epithelial cell lines and human primary podocytes. Our study revealed an increased incidence of mitotic cells with surplus microtubule-organizing centers fostering multipolar spindle assembly, leading to nuclear abnormalities, particularly multi-micronucleation. The levels of expression of exogenous pathogenic INF2 were similar to those of endogenous INF2. The aberrant nuclear phenotypes were observed regardless of the expression method used (retrovirus infection or plasmid transfection) or the promoter (LTR or CMV) used, and were absent with exogenous wild type INF2 expression. This indicates that the effect of pathogenic INF2 is not due to overexpression or experimental cell manipulation, but instead to the intrinsic properties of pathogenic INF2. Inactivation of the INF2 catalytic domain prevented aberrant nuclei formation. Pathogenic INF2 triggered the translocation of the transcriptional cofactor MRTF into the nucleus. RNA sequencing revealed a profound alteration in the transcriptome that could be primarily attributed to the sustained activation of the MRTF-SRF transcriptional complex. Cells eventually underwent mitotic catastrophe and death. Reducing MRTF-SRF activation mitigated multi-micronucleation, reducing the extent of cell death. Our results, if validated in animal models, could provide insights into the mechanism driving glomerular degeneration in INF2-linked FSGS and may suggest potential therapeutic strategies for impeding FSGS progression.

The physical and cellular mechanism of structural color change in zebrafish

Many animals exhibit remarkable colors that are produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These animals can fine-tune their crystal-based structural colors to communicate with each other, regulate body temperature, and create camouflage. While it is known that these changes in color are caused by changes in the angle of the crystal arrays relative to incident light, the cellular machinery that drives color change is not understood. Here, using a combination of 3D focused ion beam scanning electron microscopy (FIB-SEM), micro-focused X-ray diffraction, superresolution fluorescence light microscopy, and pharmacological perturbations, we characterized the dynamics and 3D cellular reorganization of crystal arrays within zebrafish iridophores during norepinephrine (NE)-induced color change. We found that color change results from a coordinated 20° tilting of the intracellular crystals, which alters both crystal packing and the angle at which impinging light hits the crystals. Importantly, addition of the dynein inhibitor dynapyrazole-a completely blocked this NE-induced red shift by hindering crystal dynamics upon NE addition. FIB-SEM and microtubule organizing center (MTOC) mapping showed that microtubules arise from two MTOCs located near the poles of the iridophore and run parallel to, and in between, individual crystals. This suggests that dynein drives crystal angle change in response to NE by binding to the limiting membrane surrounding individual crystals and walking toward microtubule minus ends. Finally, we found that intracellular cAMP regulates the color change process. Together, our results provide mechanistic insight into the cellular machinery that drives structural color change.

The kinase ZYG-1 phosphorylates the cartwheel protein SAS-5 to drive centriole assembly in C. elegans

Centrioles organize centrosomes, the cell’s primary microtubule-organizing centers (MTOCs). Centrioles double in number each cell cycle, and mis-regulation of this process is linked to diseases such as cancer and microcephaly. In C. elegans, centriole assembly is controlled by the Plk4 related-kinase ZYG-1, which recruits the SAS-5–SAS-6 complex. While the kinase activity of ZYG-1 is required for centriole assembly, how it functions has not been established. Here we report that ZYG-1 physically interacts with and phosphorylates SAS-5 on 17 conserved serine and threonine residues in vitro. Mutational scanning reveals that serine 10 and serines 331/338/340 are indispensable for proper centriole assembly. Embryos expressing SAS-5S10A exhibit centriole assembly failure, while those expressing SAS-5S331/338/340A possess extra centrioles. We show that in the absence of serine 10 phosphorylation, the SAS-5–SAS-6 complex is recruited to centrioles, but is not stably incorporated, possibly due to a failure to coordinately recruit the microtubule-binding protein SAS-4. Our work defines the critical role of phosphorylation during centriole assembly and reveals that ZYG-1 might play a role in preventing the formation of excess centrioles.

Translational control of MPS1 links protein synthesis with the initiation of cell division and spindle pole body duplication in Saccharomyces cerevisiae.

Protein synthesis underpins cell growth and controls when cells commit to a new round of cell division at a point in late G1 of the cell cycle called Start. Passage through Start also coincides with the duplication of the microtubule-organizing centers, the yeast spindle pole bodies, which will form the 2 poles of the mitotic spindle that segregates the chromosomes in mitosis. The conserved Mps1p kinase governs the duplication of the spindle pole body (SPB) in Saccharomyces cerevisiae. Here, we show that the MPS1 transcript has a short upstream open reading frame (uORF) that represses the synthesis of Mps1p. Mutating the MPS1 uORF makes the cells smaller, accelerates the appearance of Mps1p in late G1, and promotes completion of Start. Monitoring the SPB in the cell cycle using structured illumination microscopy revealed that mutating the MPS1 uORF enabled cells to duplicate their SPB earlier at a smaller cell size. The accelerated Start of MPS1 uORF mutants depends on the G1 cyclin Cln3p and the transcriptional repressor Whi5p but not on the Cln1,2p G1 cyclins. These results identify growth inputs in mechanisms that control duplication of the microtubule-organizing center and implicate these processes in the coupling of cell growth with division.

Triphenyltin chloride exposure inhibits meiotic maturation of mouse oocytes by disrupting cytoskeleton assembly and cell cycle progression

Triphenyltin chloride (TPTCL) is widely used in various industrial and agricultural applications. This study aimed to elucidate the mechanisms underlying the toxicological effects of TPTCL on oocytes. The obtained findings revealed that TPTCL exposure reduced polar body extrusion (PBE) and induced meiotic arrest. Mechanistically, TPTCL disrupted meiotic spindle assembly and chromosome alignment. Further analysis indicated a significant decrease in p-MAPK expression, and disturbances in the localization of Pericentrin and p-Aurora A in TPTCL exposed oocytes, which suggesting impaired microtubule organizing center (MTOC)function. Moreover, TPTCL exposure enhance microtubule acetylation and microtubule instability. Therefore, the spindle assembly checkpoint (SAC) remained activated, and the activity of the anaphase-promoting complex (APC) was inhibited, thereby preventing oocytes from progressing into the entering anaphase I (AI) stage. TPTCL exposure also augmented the actin filaments in the cytoplasm. Notably, mitochondrial function appeared unaffected by TPTCL, as evidenced indicated by stable mitochondrial membrane potential and ATP content. Furthermore, TPTCL treatment altered H3K27me2, H3K27me3 and H3K9me3 levels, suggesting changes in epigenetic modifications in oocytes. Taken together, our results suggest that TPTCL disrupts cytoskeleton assembly, continuously activates SAC, inhibits APC activity, and blocks meiotic progression, ultimately impair oocyte maturation.

Abstract 628: RNA binding protein Matrin3 acts as a tumor suppressor by inhibiting microtubule nucleation

Abstract Introduction: Microtubule-associated proteins (MAPs) regulate microtubule dynamics, which is critical for controlling cell division, proliferation, migration, and intracellular transport. Due to their critical role in maintaining cellular integrity, microtubules have served as potent therapeutic targets for cancer treatment. Here, we report “Matrin 3 (MATR3)” as a novel MAP that interacts with β- tubulin and γ-tubulin, which constitutes microtubule organizing center (MTOC) essential for proper spindle formation and chromosomal segregation. Methods: Breast cancer cell lines (MDA-MB-231, MDA-MB-468, SKBR3, MCF7, BT549, HMEC, and MCF10A) were purchased from the American Type Culture Collection and cultured in standard medium. Breast cancer cells were transfected either with MATR3 overexpression plasmid or siRNA specific to MATR3. These overexpression/knockdown breast cancer cells were analyzed for cell viability, migration, invasion, colony formation, cell cycle, apoptosis assays, RNA sequencing, RT-qPCR, western blotting, RNA immunoprecipitation, and in vivo tumor xenograft study. Results: Our findings show that MATR3 binds to RNA encoding MAPs including β-tubulin and γ-tubulin, and regulate their stabilization. MATR3 also interacted with β-tubulin protein. Our data demonstrate that MATR3 functions as a tumor suppressor, as its overexpression inhibits cancer growth, while its depletion increased tumor growth in vivo. Mechanistic studies confirmed that MATR3 mediates its tumor suppressor role by regulating the expression of the MTOC-associated protein-encoding gene MZT2B (Mitotic Spindle Organizing Protein 2B), which is overexpressed in cancers and promotes cancer growth. Further, loss of MATR3 expression or function results in dysregulated microtubule dynamics and uncontrolled expression of the oncogenic proteins including MZT2B. Conclusions: In conclusion, our data strongly suggests that MATR3 serves as a novel RNA-binding protein with tumor suppressor properties. This discovery holds significant importance as MATR3 becomes the second protein, following Adenomatous Polyposis Coli (APC), to be identified for its binding to both MAP RNA and protein. Citation Format: Panneerdoss Subbarayalu, Subapriya Rajamanickam, Santosh Timilsina, Saif Nirzhor, Daisy Medina, Shahad Abdulsahib, Pitta V Prabhakar, Deepika Singh, Fu-Yang Li, Pooja Yadav, Esha Reddy, Krishna Evani, Vijay Eedunuri, Trong Phat Do, Benjamin C. Onyeagucha, Tabrez A. Mohammad, Jae-Hoon Ji, Yidong Chen, Nourhan Abdelfattah, Nicholas Dybdal-Hargreaves, Li-Ju Wang, Yu-Chiao Chiu, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi, Manjeet K. Rao. RNA binding protein Matrin3 acts as a tumor suppressor by inhibiting microtubule nucleation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 628.

Abstract 1647: Plk4 phosphorylates the distal tip complex to promote centriole growth

Abstract The centrosome is the major microtubule-organizing center of the cell with a key role in forming the mitotic spindle. The centrosome consists of two core duplicating elements, the centrioles, surrounded by pericentriolar material, a cloud of proteins that nucleates microtubules. Centrioles are barrel-like structures composed of microtubule bundles and are essential to maintain centrosome number. Therefore, centriole duplication and growth are tightly regulated to ensure proper centrosome number throughout the cell cycle. Alterations in centrosome number lead to an abnormally shaped mitotic spindle that often mis-segregate chromosomes, thereby causing chromosomal instability, a hallmark of cancer. Defects in centriole growth, such as over-elongation, can increase centrosome numbers (known as ‘centriole amplification’, an overproduction of centrioles frequently observed in cancer cells). For example, over-elongation of the centriolar barrel can cause multiple ectopic daughter centrioles to assemble or simply fragment, producing multiple smaller (but functional) centrioles. Thus, understanding how centrioles grow and define length is a major question in the field. Centrioles elongate at their distal ends and, in Drosophila, elongation is regulated by a group of proteins, Cep97, CP110 and Klp10A. Elongation is negatively regulated by Cep97 and CP110 which cap centrioles at their distal end to maintain centriole length, by counteracting the microtubule depolymerizing kinesin, Klp10A. Recently, we identified a new centriole regulator, Cep104, that also localizes to the centriole distal tip. We discovered that Cep104 promotes centriole elongation through its microtubule-binding TOG domain. Additionally, Cep104 localization to the centriole distal ends is dependent on Cep97. Furthermore, Cep97, CP110, Cep104 and Klp10A form a complex and we have mapped their sites of interaction. Collectively, we refer to this as the Distal Tip Complex (DTC). Although the DTC is first recruited to new daughter centrioles during G1/S-phase, the majority of centriole elongation occurs during mitosis. Here, we explore the mechanisms that regulate the DTC to promote centriole growth during mitosis. We hypothesize that Polo-like kinase 4 (Plk4), the master regulator of centriole duplication, phosphorylates and inhibits the DTC to promote elongation of nascent centrioles. Notably, we found that centrioles are longer in cells overexpressing Plk4. Using tandem mass spectrometry, we identified multiple sites that Plk4 phosphorylates on each DTC protein. Analysis of DTC phosphomutants revealed that the phosphorylation state of Klp10A, Cep97 and CP110 affect centriole number. We are currently investigating how Plk4 phosphorylation regulates the activities of these conserved centriole length factors. Citation Format: Anastasia Amoiroglou, John M. Ryniawec, Daniel W. Buster, Gregory C. Rogers. Plk4 phosphorylates the distal tip complex to promote centriole growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1647.

Fungal microtubule organizing centers are evolutionarily unstable structures

For most Eukaryotic species the requirements of cilia formation dictate the structure of microtubule organizing centers (MTOCs). In this study we find that loss of cilia corresponds to loss of evolutionary stability for fungal MTOCs. We used iterative search algorithms to identify proteins homologous to those found in Saccharomyces cerevisiae, and Schizosaccharomyces pombe MTOCs, and calculated site-specific rates of change for those proteins that were broadly phylogenetically distributed. Our results indicate that both the protein composition of MTOCs as well as the sequence of MTOC proteins are poorly conserved throughout the fungal kingdom. To begin to reconcile this rapid evolutionary change with the rigid structure and essential function of the S. cerevisiae MTOC we further analyzed how structural interfaces among proteins influence the rates of change for specific residues within a protein. We find that a more stable protein may stabilize portions of an interacting partner where the two proteins are in contact. In summary, while the protein composition and sequences of the MTOC may be rapidly changing the proteins within the structure have a stabilizing effect on one another. Further exploration of fungal MTOCs will expand our understanding of how changes in the functional needs of a cell have affected physical structures, proteomes, and protein sequences throughout fungal evolution.

The fission yeast NDR kinase Orb6 and its signalling pathway MOR regulate cytoplasmic microtubule organization during the cell cycle.

Microtubule organization and reorganization during the cell cycle are achieved by regulation of the number, distribution and activity of microtubule-organizing centres (MTOCs). In fission yeast, the Mto1/2 complex determines the activity and distribution of cytoplasmic MTOCs. Upon mitosis, cytoplasmic microtubule nucleation ceases; inactivation of the Mto1/2 complex is triggered by Mto2 hyperphosphorylation. However, the protein kinase(s) that phosphorylates Mto2 remains elusive. Here we show that a conserved signalling network, called MOR (morphogenesis Orb6 network) in fission yeast, negatively regulates cytoplasmic MTOCs through Mto2 phosphorylation to ensure proper microtubule organization. Inactivation of Orb6 kinase, the most downstream MOR component, by attenuation of MOR signalling leads to reduced Mto2 phosphorylation, coincident with increased number of both Mto2 puncta and cytoplasmic microtubules. These defects cause the emergence of uncoordinated mitotic cells with cytoplasmic microtubules, resulting in reduced spindle assembly. Thus, the regulation of Mto2 by the MOR is crucial for cytoplasmic microtubule organization and contributes to reorganization of the microtubule cytoskeletons during the cell cycle.

CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

γ-Tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends but not the minus-end tracking protein CAMSAP1, induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits the formation of CAMSAP2-bound microtubules detached from the microtubule-organizing centre. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.

β-catenin mediates growth defects induced by centrosome loss in a subset of APC mutant colorectal cancer independently of p53.

Colorectal cancer is the third most common cancer and the second leading cause of cancer-related deaths worldwide. The centrosome is the main microtubule-organizing center in animal cells and centrosome amplification is a hallmark of cancer cells. To investigate the importance of centrosomes in colorectal cancer, we induced centrosome loss in normal and cancer human-derived colorectal organoids using centrinone B, a Polo-like kinase 4 (Plk4) inhibitor. We show that centrosome loss represses human normal colorectal organoid growth in a p53-dependent manner in accordance with previous studies in cell models. However, cancer colorectal organoid lines exhibited different sensitivities to centrosome loss independently of p53. Centrinone-induced cancer organoid growth defect/death positively correlated with a loss of function mutation in the APC gene, suggesting a causal role of the hyperactive WNT pathway. Consistent with this notion, β-catenin inhibition using XAV939 or ICG-001 partially prevented centrinone-induced death and rescued the growth two APC-mutant organoid lines tested. Our study reveals a novel role for canonical WNT signaling in regulating centrosome loss-induced growth defect/death in a subset of APC-mutant colorectal cancer independently of the classical p53 pathway.

BGLF4 kinase regulates the formation of the EBV cytoplasmic assembly compartment and the recruitment of cellular IQGAP1 for virion release.

After Epstein-Barr virus (EBV) genome replication and encapsidation in the nucleus, nucleocapsids are translocated into the cytoplasm for subsequent tegumentation and maturation. The EBV BGLF4 kinase, which induces partial disassembly of the nuclear lamina, and the nuclear egress complex BFRF1/BFLF2 coordinately facilitate the nuclear egress of nucleocapsids. Here, we demonstrate that within EBV reactivated epithelial cells, viral capsids, tegument proteins, and glycoproteins are clustered in the juxtanuclear concave region, accompanied by redistributed cytoplasmic organelles and the cytoskeleton regulator IQ-domain GTPase-activation protein 1 (IQGAP1), close to the microtubule-organizing center (MTOC). The assembly compartment (AC) structure was diminished in BGLF4-knockdown TW01-EBV cells and BGLF4-knockout bacmid-carrying TW01 cells, suggesting that the formation of AC structure is BGLF4-dependent. Notably, glycoprotein gp350/220 was observed by confocal imaging to be distributed in the perinuclear concave region and surrounded by the endoplasmic reticulum (ER) membrane marker calnexin, indicating that the AC may be located within a globular structure derived from ER membranes, adjacent to the outer nuclear membrane. Moreover, the viral capsid protein BcLF1 and tegument protein BBLF1 were co-localized with IQGAP1 near the cytoplasmic membrane in the late stage of replication. Knockdown of IQGAP1 did not affect the AC formation but decreased virion release from both TW01-EBV and Akata+ cells, suggesting IQGAP1-mediated trafficking regulates EBV virion release. The data presented here show that BGLF4 is required for cytoskeletal rearrangement, coordination with the redistribution of cytoplasmic organelles and IQGAP1 for virus maturation, and subsequent IQGAP1-dependent virion release.IMPORTANCEEBV genome is replicated and encapsidated in the nucleus, and the resultant nucleocapsids are translocated to the cytoplasm for subsequent virion maturation. We show that a cytoplasmic AC, containing viral proteins, markers of the endoplasmic reticulum, Golgi, and endosomes, is formed in the juxtanuclear region of epithelial and B cells during EBV reactivation. The viral BGLF4 kinase contributes to the formation of the AC. The cellular protein IQGAP1 is also recruited to the AC and partially co-localizes with the virus capsid protein BcLF1 and tegument protein BBLF1 in EBV-reactivated cells, dependent on the BGLF4-induced cytoskeletal rearrangement. In addition, virion release was attenuated in IQGAP1-knockdown epithelial and B cells after reactivation, suggesting that IQGAP1-mediated trafficking may regulate the efficiency of virus maturation and release.

Programmed disassembly of a microtubule-based membrane protrusion network coordinates 3D epithelial morphogenesis in Drosophila.

Comprehensive analysis of cellular dynamics during the process of morphogenesis is fundamental to understanding the principles of animal development. Despite recent advancements in light microscopy, how successive cell shape changes lead to complex three-dimensional tissue morphogenesis is still largely unresolved. Using in vivo live imaging of Drosophila wing development, we have studied unique cellular structures comprising a microtubule-based membrane protrusion network. This network, which we name here the Interplanar Amida Network (IPAN), links the two wing epithelium leaflets. Initially, the IPAN sustains cell-cell contacts between the two layers of the wing epithelium through basal protrusions. Subsequent disassembly of the IPAN involves loss of these contacts, with concomitant degeneration of aligned microtubules. These processes are both autonomously and non-autonomously required for mitosis, leading to coordinated tissue proliferation between two wing epithelia. Our findings further reveal that a microtubule organization switch from non-centrosomal to centrosomal microtubule-organizing centers (MTOCs) at the G2/M transition leads to disassembly of non-centrosomal microtubule-derived IPAN protrusions. These findings exemplify how cell shape change-mediated loss of inter-tissue contacts results in 3D tissue morphogenesis.