Control Of Microtubule Dynamics Research Articles

Abstract 3157: The crosstalk between STAT3 and p53/ERK signaling regulates brain metastasis

Abstract Brain metastasis (BM) affects approximately 10% of cancer patients with metastatic disease, representing a major cause of morbidity and mortality. BM is a complex multi-step process involving various immune checkpoint proteins. Mitogen-activated protein kinase (MAPK), particularly extracellular signal-regulated kinases 1/2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3) are implicated in tumorigenesis and are critical upstream regulators of Programmed Death Ligand 1 (PD-L1) expression, a common immunotherapy target. Tumor suppressor p53 is shown to regulate STAT3 and ERK1/2 signaling, which are dysregulated in various malignancies. We hypothesize that p53 along with STAT3 and ERK1/2 play a critical role in the initiation and maintenance of BM. Twenty-six BM tumors with various primary sites of origin were used following IRB guidelines. pSTAT3Tyr705, pERK1/2Thr202/Tyr204, mutant p53 (p53mt), and PD-L1 expression was determined by immunohistochemistry. Gene expression analysis of BM was performed on a cDNA microarray. Human brain metastatic breast cancer cells (MDA-MB-231) were treated with STAT3 inhibitor (NSC74859) or MAPK inhibitor (U0126) in regular or astrocytic media to determine cell proliferation and migration by MTT assay and scratch-wound, respectively. ERK1/2 signaling pathway status was determined by Western blotting. Results demonstrated: 1) pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 were expressed profusely at tumor margins, while p53mt was uniformly expressed throughout. 81% of BM expressed p53mt while 86% and 63% expressed pERK1/2Thr202/Tyr204 and pSTAT3Tyr705, respectively. Venn analysis displayed significant overlap between expression, with 62% overlap between p53mt and pERK1/2Thr202/Tyr204 and a 50% overlap between both pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 with p53mt. Expression of PD-L1 was seen in a majority of BM tumors. 2) Gene expression analysis demonstrated alterations in 32 STAT3- and MAPK-associated genes; these included upregulation of stathmin-1 and stathmin-like 3, which control microtubule dynamics in STAT3-related cell migration, and changes in cytokine-related genes, suggesting altered immune responses. 3) Cell proliferation showed augmented growth in astrocytic media (p≤0.05) and was significantly decreased following treatment with NSC74850 or U0126 (p≤0.01). While migration showed a trend towards significance in reduction following NSC74859 (p=0.08), migration stagnated following U0126 treatment in astrocytic media (p≤0.01). 4) pERK1/2Thr202/Tyr204 expression was reduced following U0126 or NSC74859 treatment. These findings suggest activation of STAT3 and ERK1/2 promote BM and suppression of these pathways may lead to a reduction in cell viability and motility. Therefore, this provides compelling evidence for the use of STAT3 and ERK1/2 along with p53 as potential targets for immunotherapy in brain metastatic disease. Citation Format: Sabrina L. Zeller, Eris Spirollari, John V. Wainwright, Simon J. Hanft, Chirag D. Gandhi, Meena Jhanwar-Uniyal. The crosstalk between STAT3 and p53/ERK signaling regulates brain metastasis [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 3157.

A photo-SAR study of photoswitchable azobenzene tubulin-inhibiting antimitotics identifying a general method for near-quantitative photocontrol.

Azobenzene analogues of the tubulin polymerisation inhibitor combretastatin A4 (PSTs) were previously developed to optically control microtubule dynamics in living systems, with subsecond response time and single-cell spatial precision, by reversible in situ photoswitching of their bioactivity with near-UV/visible light. First-generation PSTs were sufficiently potent and photoswitchable for use in live cells and embryos. However, the link between their seconds-scale and hours-scale bioactivity remained untested. Furthermore, the scope for modifications to tune their photo-structure-activity-relationship or expand their function was unknown. Here, we used large-field-of-view, long-term tandem photoswitching/microscopy to reveal the temporal onset of cytostatic effects. We then synthesised a panel of novel PSTs exploring structural variations that tune photoresponse wavelengths and lipophilicity, identifying promising blue-shifted analogues that are better-compatible with GFP/YFP imaging. Taken together, these results can guide new design and applications for photoswitchable microtubule inhibitors. We also identified tolerated sites for linkers to attach functional cargos; and we tested fluorophores, aiming at RET isomerisation or reporter probes. Instead we found that these antennas greatly enhance long-wavelength single-photon photoisomerisation, by an as-yet un-explored mechanism, that can now drive general progress towards near-quantitative long-wavelength photoswitching of photopharmaceuticals in living systems, with minimal molecular redesign and broad scope.

Neurofilaments in health and Charcot-Marie-Tooth disease.

Neurofilaments (NFs) are the most abundant component of mature neurons, that interconnect with actin and microtubules to form the cytoskeleton. Specifically expressed in the nervous system, NFs present the particularity within the Intermediate Filament family of being formed by four subunits, the neurofilament light (NF-L), medium (NF-M), heavy (NF-H) proteins and α-internexin or peripherin. Here, we review the current knowledge on NF proteins and neurofilaments, from their domain structures and their model of assembly to the dynamics of their transport and degradation along the axon. The formation of the filament and its behaviour are regulated by various determinants, including post-transcriptional (miRNA and RBP proteins) and post-translational (phosphorylation and ubiquitination) modifiers. Altogether, the complex set of modifications enable the neuron to establish a stable but elastic NF array constituting the structural scaffold of the axon, while permitting the local expression of NF proteins and providing the dynamics necessary to fulfil local demands and respond to stimuli and injury. Thus, in addition to their roles in mechano-resistance, radial axonal outgrowth and nerve conduction, NFs control microtubule dynamics, organelle distribution and neurotransmission at the synapse. We discuss how the studies of neurodegenerative diseases with NF aggregation shed light on the biology of NFs. In particular, the NEFL and NEFH genes are mutated in Charcot-Marie-Tooth (CMT) disease, the most common inherited neurological disorder of the peripheral nervous system. The clinical features of the CMT forms (axonal CMT2E, CMT2CC; demyelinating CMT1F; intermediate I-CMT) with symptoms affecting the central nervous system (CNS) will allow us to further investigate the physiological roles of NFs in the brain. Thus, NF-CMT mouse models exhibit various degrees of sensory-motor deficits associated with CNS symptoms. Cellular systems brought findings regarding the dominant effect of NF-L mutants on NF aggregation and transport, although these have been recently challenged. Neurofilament detection without NF-L in recessive CMT is puzzling, calling for a re-examination of the current model in which NF-L is indispensable for NF assembly. Overall, we discuss how the fundamental and translational fields are feeding each-other to increase but also challenge our knowledge of NF biology, and to develop therapeutic avenues for CMT and neurodegenerative diseases with NF aggregation.

High Tumoral STMN1 Expression Is Associated with Malignant Potential and Poor Prognosis in Patients with Neuroblastoma.

Stathmin 1 (STMN1), a marker for immature neurons and tumors, controls microtubule dynamics by destabilizing tubulin. It plays an essential role in cancer progression and indicates poor prognosis in several cancers. This potential protein has not been clarified in clinical patients with neuroblastoma. Therefore, this study aimed to assess the clinical significance and STMN1 function in neuroblastoma with and without MYCN amplification. Using immunohistochemical staining, STMN1 expression was examined in 81 neuroblastoma samples. Functional analysis revealed the association among STMN1 suppression, cellular viability, and endogenous or exogenous MYCN expression in neuroblastoma cell lines. High levels of STMN1 expression were associated with malignant potential, proliferation potency, and poor prognosis in neuroblastoma. STMN1 expression was an independent prognostic factor in patients with neuroblastoma. Furthermore, STMN1 knockdown inhibited neuroblastoma cell growth regardless of endogenous and exogenous MYCN overexpression. Our data suggest that assessing STMN1 expression in neuroblastoma could be a powerful indicator of prognosis and that STMN1 might be a promising therapeutic candidate against refractory neuroblastoma with and without MYCN amplification.

The kinesin Kif21b regulates radial migration of cortical projection neurons through a non-canonical function on actin cytoskeleton

Completion of neuronal migration is critical for brain development. Kif21b is a plus-end-directed kinesin motor protein that promotes intracellular transport and controls microtubule dynamics in neurons. Here we report a physiological function of Kif21b during radial migration of projection neurons in the mouse developing cortex. Invivo analysis in mouse and live imaging on cultured slices demonstrate that Kif21b regulates the radial glia-guided locomotion of newborn neurons independently of its motility on microtubules. We show that Kif21b directly binds and regulates the actin cytoskeleton both invitro and invivo in migratory neurons. We establish that Kif21b-mediated regulation of actin cytoskeleton dynamics influences branching and nucleokinesis during neuronal locomotion. Altogether, our results reveal atypical roles of Kif21b on the actin cytoskeleton during migration of cortical projection neurons.

Lisavanbulin in patients with recurrent glioblastoma: Phase 2a results and a consolidated analysis of response-predictive biomarkers.

2044 Background: Lisavanbulin (BAL101553, prodrug of BAL27862) destabilizes microtubules and promotes tumor cell death by modulating the spindle assembly checkpoint. BAL27862 is a lipophilic small molecule shown in rodents to penetrate the brain, with antitumor activity in orthotopic glioblastoma (GBM) models. In the Phase 1 part of this study1 (NCT02490800), 2 of 5 patients with recurrent IDH-mutated G4 astrocytoma treated at active dose levels (15–30 mg/day) showed long-lasting objective responses and strong end-binding protein 1 (EB1) expression in GBM tissue by IHC. EB1 is a regulator protein on microtubules. Methods: The objective of the Phase 2 study was to investigate prospectively the response-predictive value of EB1, and to identify RNA-based response signatures in patients with recurrent GBM. A Simon’s two-stage design was used with an objective response rate (ORR) ≥ 2/9 required in Stage 1 to enable a final ORR ≥ 6/19. A prescreening program identified patients with EB1-positive archival GBM tissue. All patients received 25 mg oral lisavanbulin once daily. RNA-seq was performed on archival GBM tissues. Results: GBM tissue samples from 64 of 629 patients (10.2%) obtained from 13 sites in four countries were EB1-positive, and 18 of these patients received lisavanbulin. Of 9 patients with measurable disease evaluable for response in Stage 1, one patient had a partial response, and another had a 44% target lesion area reduction. Despite sustained activity in these patients, formal stage transition criteria were not met, and the study was closed. IHC testing for EB1 did not show sufficient enrichment for response, but it is thought that IDH status may play a role. In addition, RNA-seq analyses identified a five-gene signature that is distinct from expression patterns observed with EB1-IHC positivity, and predicts current responses irrespective of IDH status. This signature is characterized by homeobox gene downregulation, which may be implicated in the control of microtubule dynamics. Conclusions: This Phase 2a study supports previous study results that lisavanbulin is associated with durable responses and clinical benefit in a subset of patients with GBM. RNA-seq analyses of GBM samples suggest further evaluation of the lisavanbulin predictive response signature. 1Lopez et al, JCO 2019;37,15 suppl, 2025. Clinical trial information: NCT02490800 . [Table: see text]

Abstract 4896: NF1 (neurofibromatosis 1) controls microtubule dynamics and dictates sensitivity to maytansinoids

Abstract The tumor suppressor NF1 is classically considered a negative RAS regulator, but sparse evidence suggests additional RAS-independent roles. Early studies suggested an interaction with tubulin, which remains poorly characterized to date but may be of particular therapeutic interest as NF1 is somatically mutated across multiple tumor types. We showed that multiple CRISPR-Cas9-engineeerd NF1 KO HER2+ breast cancer cells (BT-474, SK-BR3, HCC1954) become exquisitely sensitive to the Antibody-Drug Conjugate (ADC) Trastuzumab emtansine (T-DM1); we here investigate the underlying mechanism.TDM1 hypersensitivy was specific to the maytansin microtubule-targeting payload, since it was i) replicated by the naked payload but not the naked antibody; ii) absent with other ADCs (T-DxD); iii) not accompanied by increased TDM1 uptake; iv) associated with increased tubulin-maytansin binding in KO cells, as per Cellular Thermal Shift Assay. The mechanism is likely RAS-independent, as KRAS G12V-overexpression did not alter TDM1 sensitivity. RNAseq revealed that KO cells deregulated genes associated with microtubular dynamics and G2-M transition more strongly upon TDM1-treatement. Multiple KO cells investigated by static and live imaging exhibited marked signs of altered mitosis, with longer G2/M, supernumerary centrosomes, chromosome misalignment and frequent aneuploidy, which could also be inferred in multiple public sequencing datasets (TCGA, MSK IMPACT, AACR GENIE). Based on this, we explored NF1 role on microtubule dynamics. We found several lines of evidence for a direct and mitosis-selective interaction between NF1 and tubulin: i) in Immunofluorescence (IF), NF1 was upregulated in mitosis and colocalized with the mitotic spindle; ii) NF1 co-Immunoprecipitated with tubulin in mitosis-enriched but not asynchronous cells; iii) purified NF1 co-eluted with tubulin in size-selection chromatography; iv) in silico modeling with AlphaFold2 predicted an interaction between NF1 central domains and the alpha-beta tubulin dimer. Crucially, KO cells exhibited severe microtubule hypodynamism in cold-induced depolymerization-repolymerization assays, replicated in multiple cell types and accompanied by imbalanced levels of plus/minus-end microtubule-associated proteins. By IF on cocultured WT/KO live cells, KO cells showed significantly higher GTP-tubulin, known to cause microtubular hyperstability, suggesting the intriguing possibility that NF1 may directly regulate tubulin intrinsic GTP-hydrolyzing activity, similar to its role on RAS. In conclusion, we provide extensive mechanistic evidence for a direct and previously underappreciated role of NF1 in microtubular dynamics, which reshapes our understanding of its tumor-suppressive activity and provides a rationale for pharmacological targeting of NF1-mutated tumors. Citation Format: Bruno A. Duso, Eleonora Messuti, Emanuele Bonetti, Giulia Tini, Alessia Castiglioni, Giuseppe Ciossani, Silvia Monzani, Chiara Soriani, Simona Rodighiero, Luigi Scietti, Costantino Jemos, Luca Mazzarella. NF1 (neurofibromatosis 1) controls microtubule dynamics and dictates sensitivity to maytansinoids. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4896.

Boosting ferroptosis and microtubule inhibition for antitumor therapy via a carrier-free supermolecule nanoreactor

Traditional microtubule inhibitors fail to significantly enhance the effect of colorectal cancer; hence, new and efficient strategies are necessary. In this study, a supramolecular nanoreactor (DOC@TA-Fe3+) based on tannic acid (TA), iron ion (Fe3+), and docetaxel (DOC) with microtubule inhibition, reactive oxygen species (ROS) generation, and glutathione peroxidase 4 (GPX4) inhibition, is prepared for ferroptosis/apoptosis treatment. After internalization by CT26 cells, the DOC@TA-Fe3+ nanoreactor escapes from the lysosomes to release payloads. The subsequent Fe3+/Fe2+ conversion mediated by TA reducibility can trigger the Fenton reaction to enhance the ROS concentration. Additionally, Fe3+ can consume glutathione to repress the activity of GPX4 to induce ferroptosis. Meanwhile, the released DOC controls microtubule dynamics to activate the apoptosis pathway. The superior in vivo antitumor efficacy of DOC@TA-Fe3+ nanoreactor in terms of tumor growth inhibition and improved survival is verified in CT26 tumor-bearing mouse model. Therefore, the nanoreactor can act as an effective apoptosis and ferroptosis inducer for application in colorectal cancer therapy.

MAPRE2 regulates the first meiotic progression in mouse oocytes

Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, cell migration and morphogenesis. Microtubule-associated RP/EB family member 2 (MAPRE2/EB2) is a highly conserved core component of +TIPs networks, but whether this molecule is required for mammalian meiotic progression is unknown. In this study, we investigated the expression and function of MAPRE2 during oocyte maturation. Our results showed that MAPRE2 was consistently expressed from germinal vesicle (GV) to metaphase II (MII) stages and that MAPRE2 was distributed in the cytoplasm of oocytes at GV stage and along the spindle at metaphase I (MI) and MII stages. Small interfering RNA-mediated knockdown of Mapre2 severely impaired microtubule stability, kinetochore-microtubule attachment, and chromosome alignment and subsequently caused spindle assembly checkpoint (SAC) activation and cyclin B1 nondegradation, leading to failure of chromosome segregation and first polar body extrusion. This study demonstrates for the first time that MAPRE2 plays an important role during mouse oocyte meiosis.

Stathmins and Motor Neuron Diseases: Pathophysiology and Therapeutic Targets.

Motor neuron diseases (MNDs) are a group of fatal, neurodegenerative disorders with different etiology, clinical course and presentation, caused by the loss of upper and lower motor neurons (MNs). MNs are highly specialized cells equipped with long, axonal processes; axonal defects are some of the main players underlying the pathogenesis of these disorders. Microtubules are key components of the neuronal cytoskeleton characterized by dynamic instability, switching between rapid polymerization and shrinkage. Proteins of the stathmin family affect microtubule dynamics regulating the assembly and the dismantling of tubulin. Stathmin-2 (STMN2) is one of the most abundantly expressed genes in MNs. Following axonal injury, STMN2 expression is upregulated, and the protein is transported toward the growth cones of regenerating axons. STMN2 has a critical role in axonal maintenance, and its dysregulation plays an important role in neurodegenerative processes. Stathmin-1 (STMN1) is a ubiquitous protein that is highly expressed during the development of the nervous system, and its phosphorylation controls microtubule dynamics. In the present review, we summarize what is currently known about the involvement of stathmin alterations in MNDs and the potential therapeutic effect of their modulation, with a specific focus on the most common forms of MND, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA).

Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules

Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex.1,2 In mammalian cells, end-binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for diverse+TIPs that control microtubule dynamics and interactions with other intracellular structures.3 Because binding of many+TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation,4-6 the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated+TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light-sensitive EB1 variant, π-EB1, that allows local, acute, and reversible inactivation of+TIP association with growing microtubule ends in live cells.7 We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase but instead increases astral microtubule length and number. Yet in the absence of EB1 activity, astral microtubules fail to engage the cortical dynein/dynactin machinery, and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments, likely due to compensatory molecular systems regulating vertebrate spindle dynamics.

Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents.

Microtubule dynamics can be inhibited with sub-second temporal resolution and cellular-scale spatial resolution, by using precise illuminations to optically pattern where and when photoswitchable microtubule-inhibiting chemical reagents exert their latent bioactivity. The recently available reagents (SBTub, PST, STEpo, AzTax, PHTub) now enable researchers to use light to reversibly modulate microtubule-dependent processes in eukaryotes, in 2D and 3D cell culture as well as in vivo, across a variety of model organisms: with applications in fields from cargo transport to cell migration, cell division, and embryonic development.Here we give an introduction to using these photoswitchable microtubule inhibitors in cells. We describe the theory of small molecule photoswitching, andthe unique performance features, usage requirements, and limitations that photoswitchable chemical reagents have; then we summarize the major classes of photoswitchable microtubule inhibitors that are currently available, withthe properties that suit them to different applications, and troubleshooting measures for avoiding common mistakes. We outline workflows to establish cellular assays where they are used to optically control microtubule dynamics in a temporally reversible fashion with spatial specificity down to a single selected cell within a field of view. The methods in this chapter also equip the reader to tackle advanced uses of photoswitchable chemical reagents, in 3D culture and in vivo.

Lattice defects induced by microtubule-stabilizing agents exert a long-range effect on microtubule growth by promoting catastrophes

Microtubules are dynamic cytoskeletal polymers that spontaneously switch between phases of growth and shrinkage. The probability of transitioning from growth to shrinkage, termed catastrophe, increases with microtubule age, but the underlying mechanisms are poorly understood. Here, we set out to test whether microtubule lattice defects formed during polymerization can affect growth at the plus end. To generate microtubules with lattice defects, we used microtubule-stabilizing agents that promote formation of polymers with different protofilament numbers. By employing different agents during nucleation of stable microtubule seeds and the subsequent polymerization phase, we could reproducibly induce switches in protofilament number and induce stable lattice defects. Such drug-induced defects led to frequent catastrophes, which were not observed when microtubules were grown in the same conditions but without a protofilament number mismatch. Microtubule severing at the site of the defect was sufficient to suppress catastrophes. We conclude that structural defects within the microtubule lattice can exert effects that can propagate over long distances and affect the dynamic state of the microtubule end.

LIM Kinases in Osteosarcoma Development.

Tumorigenesis is a long-term and multistage process that often leads to the formation of metastases. During this pathological course, two major events appear to be crucial: primary tumour growth and metastatic expansion. In this context, despite research and clinical advances during the past decades, bone cancers remain a leading cause of death worldwide among paediatric cancer patients. Osteosarcomas are the most common malignant bone tumours in children and adolescents. Notwithstanding advances in therapeutic treatments, many patients succumb to these diseases. In particular, less than 30% of patients who demonstrate metastases at diagnosis or are poor responders to chemotherapy survive 5 years after initial diagnosis. LIM kinases (LIMKs), comprising LIMK1 and LIMK2, are common downstream effectors of several signalization pathways, and function as a signalling node that controls cytoskeleton dynamics through the phosphorylation of the cofilin family proteins. In recent decades, several reports have indicated that the functions of LIMKs are mainly implicated in the regulation of actin microfilament and the control of microtubule dynamics. Previous studies have thus identified LIMKs as cancer-promoting regulators in multiple organ cancers, such as breast cancer or prostate cancer. This review updates the current understanding of LIMK involvement in osteosarcoma progression.

Tracheal motile cilia in mice require CAMSAP3 for the formation of central microtubule pair and coordinated beating.

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body (BB), which are linked by a “transition zone” (TZ). The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a protein that can stabilize the minus-end of a microtubule, concentrates at multiple sites of the cilium–BB complex, including the upper region of the TZ or the axonemal basal plate (BP) where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the BP, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme and thereby supports the coordinated motion of multicilia in airway epithelial cells.

Human Cytomegalovirus Exploits TACC3 To Control Microtubule Dynamics and Late Stages of Infection.

While it is well established that microtubules (MTs) facilitate various stages of virus replication, how viruses actively control MT dynamics and functions remains less well understood. Recent work has begun to reveal how several viruses exploit End-Binding (EB) proteins and their associated microtubule plus-end tracking proteins (+TIPs), in particular to enable loading of viral particles onto MTs for retrograde transport during early stages of infection. Distinct from other viruses studied to date, at mid- to late stages of its unusually protracted replication cycle, human cytomegalovirus (HCMV) increases the expression of all three EB family members. This occurs coincident with the formation of a unique structure, termed the assembly compartment (AC), which serves as a Golgi-derived MT organizing center. Together, the AC and distinct EB proteins enable HCMV to increase the formation of dynamic and acetylated microtubule subsets to regulate distinct aspects of the viral replication cycle. Here, we reveal that HCMV also exploits EB-independent +TIP pathways by specifically increasing the expression of transforming acidic coiled coil protein 3 (TACC3) to recruit the MT polymerase, chTOG, from initial sites of MT nucleation in the AC out into the cytosol, thereby increasing dynamic MT growth. Preventing TACC3 increases or depleting chTOG impaired MT polymerization, resulting in defects in early versus late endosome organization in and around the AC as well as defects in viral trafficking and spread. Our findings provide the first example of a virus that actively exploits EB-independent +TIP pathways to regulate MT dynamics and control late stages of virus replication. IMPORTANCE Diverse viruses rely on host cell microtubule networks to transport viral particles within the dense cytoplasmic environment and to control the broader architecture of the cell to facilitate their replication. However, precisely how viruses regulate the dynamic behavior and function of microtubule filaments remains poorly defined. We recently showed that the assembly compartment (AC) formed by human cytomegalovirus (HCMV) acts as a Golgi-derived microtubule organizing center. Here, we show that at mid- to late stages of infection, HCMV increases the expression of transforming acidic coiled coil protein 3 (TACC3) to control the localization of the microtubule polymerase, chTOG. This, in turn, enables HCMV to generate dynamic microtubule subsets that organize endocytic vesicles in and around the AC and facilitate the transport of new viral particles released into the cytosol. Our findings reveal the first instance of viral targeting of TACC3 to control microtubule dynamics and virus spread.

Spastin interacts with CRMP5 to promote spindle organization in mouse oocytes by severing microtubules.

Microtubule-severing protein (MTSP) is critical for the survival of both mitotic and postmitotic cells. However, the study of MTSP during meiosis of mammalian oocytes has not been reported. We found that spastin, a member of the MTSP family, was highly expressed in oocytes and aggregated in spindle microtubules. After knocking down spastin by specific siRNA, the spindle microtubule density of meiotic oocytes decreased significantly. When the oocytes were cultured in vitro, the oocytes lacking spastin showed an obvious maturation disorder. Considering the microtubule-severing activity of spastin, we speculate that spastin on spindles may increase the number of microtubule broken ends by severing the microtubules, therefore playing a nucleating role, promoting spindle assembly and ensuring normal meiosis. In addition, we found the colocalization and interaction of collapsin response mediator protein 5 (CRMP5) and spastin in oocytes. CRMP5 can provide structural support and promote microtubule aggregation, creating transportation routes, and can interact with spastin in the microtubule activity of nerve cells (30). Knocking down CRMP5 may lead to spindle abnormalities and developmental disorders in oocytes. Overexpression of spastin may reverse the abnormal phenotype caused by the deletion of CRMP5. In summary, our data support a model in which the interaction between spastin and CRMP5 promotes the assembly of spindle microtubules in oocytes by controlling microtubule dynamics, therefore ensuring normal meiosis.

The synaptic life of microtubules.

In neurons, control of microtubule dynamics is required for multiple homeostatic and regulated activities. Over the past few decades, a great deal has been learned about the role of the microtubule cytoskeleton in axonal and dendritic transport, with a broad impact on neuronal health and disease. However, significantly less attention has been paid to the importance of microtubule dynamics in directly regulating synaptic function. Here, we review emerging literature demonstrating that microtubules enter synapses and control central aspects of synaptic activity, including neurotransmitter release and synaptic plasticity. The pleiotropic effects caused by a dysfunctional synaptic microtubule cytoskeleton may thus represent a key point of vulnerability for neurons and a primary driver of neurological disease.

DIAPH1 regulates chromosomal instability of cancer cells by controlling microtubule dynamics

Chromosomal instability (CIN) is a hallmark of cancer, resulting from misalignment and missegregation of chromosomes during meta- and anaphase, due to non-precise regulation of spindle-MT dynamics. Diaphanous Related Formin 1 (DIAPH1) is an actin nucleator and also binds microtubule (MT) with high affinity. In this study, we analyzed the role of DIAPH1 in regulation of spindle MT-dynamics and CIN in HT29 and HCT-116 colorectal cancer (CRC) cells. Our data show that down-regulation of DIAPH1 in these cell lines decreased spindle-MT speed by 50 % and the fraction of cells with misaligned and missegregated chromosomes was significantly increased. Furthermore, in HCT-116 DIAPH1 depleted cells deviation of chromosome number was elevated and the number of cells with micronuclei and cytosolic DNA was increased in both DIAPH1-knock down cell lines. In line with these results, database analysis revealed a significant correlation with low DIAPH1 mRNA expression and aneuploidy. Thus, DIAPH1 is substantially involved in the control of CIN in CRC cells. Since in vitro, DIAPH1 directly increased MT-polymerization, we assume that DIAPH1 controls CIN by regulating spindle-MT dynamics.

Disruption of the integrin-linked kinase (ILK) pseudokinase domain affects kidney development in mice

Disruption of the integrin-linked kinase (ILK) pseudokinase domain affects kidney development in mice