Modulate Microtubule Stability Research Articles

ARHGEF5 binds Drebrin and affects α-tubulin acetylation to direct neuronal morphogenesis and migration during mouse brain development.

Rho guanine nucleotide exchange factors (Rho GEFs) activate Rho GTPases, which act as molecular switches regulating various essential cellular functions. This study investigated the role of ARHGEF5, a Rho GEF known for its involvement in cell migration and invasion processes, in the context of brain development. We found that ARHGEF5 is essential for dendrite development during the early stages of neuronal growth. We also discovered that ARHGEF5 binds to Drebrin E, which is vital for coordinating actin and microtubule dynamics, and facilitates the interaction between Drebrin E and Cyclin-dependent kinase 5, which phosphorylates Drebrin E. Notably, ARHGEF5 deficiency resulted in a decrease in acetylated α-tubulin levels, and the expression of an α-tubulin acetylation mimetic mutant (K40Q) rescued the defects in dendrite development and neuronal migration, suggesting ARHGEF5's role in modulating microtubule stability. Additionally, ARHGEF5 was shown to influence Golgi positioning in the leading processes of migrating cortical neurons during brain development. Our study suggests that ARHGEF5 plays a crucial role in integrating cytoskeletal dynamics with neuronal morphogenesis and migration processes during brain development.

The cotton protein GhIQD21 interacts with GhCaM7 and modulates organ morphogenesis in Arabidopsis by influencing microtubule stability.

GhIQD21, a cotton IQ67-domain protein, interacts with GhCaM7 and alters organ shape in Arabidopsis by modulating microtubule stability. Calcium ion (Ca2+) and the calcium sensor calmodulin play crucial roles in the growth and development of plants. GhCaM7, a calmodulin in upland cotton (Gossypium hirsutum L.), is highly expressed in cotton fiber cells during the rapid elongation period and plays an important role in fiber cell development. In this study, we screened for GhCaM7-interacting proteins and identified GhIQD21, which contains a typical IQ67-domain. GhIQD21 was preferentially expressed at the fiber rapid elongation stage, and the protein localized to microtubules (MTs). Ectopic expression of GhIQD21 in Arabidopsis resulted in shorter leaves, petals, siliques, and plant height, thicker inflorescences, and more trichomes when compared with wild type (WT). Further investigation indicated that the morphogenesis of leaf epidermal cells and silique cells was altered. There was less consistency in the orientation of cortical microtubules in cotyledon and hypocotyl epidermal cells. Furthermore, compared with WT, transgenic seedling hypocotyls were more sensitive to oryzalin, a MT depolymerization drug. These results indicated that GhIQD21 is a GhCaM7-interacting protein located in MTs and that it plays a role in plant growth and potentially cotton fiber development. This study provides a foundation for further studies of the function and regulatory mechanism of GhIQD21 in fiber cell development.

The E3 ligase MREL57 modulates microtubule stability and stomatal closure in response to ABA

Regulation of stomatal movement is critical for plant adaptation to environmental stresses. The microtubule cytoskeleton undergoes disassembly, which is critical for stomatal closure in response to abscisic acid (ABA). However, the mechanism underlying this regulation largely remains unclear. Here we show that a ubiquitin-26S proteasome (UPS)-dependent pathway mediates microtubule disassembly and is required for ABA-induced stomatal closure. Moreover, we identify and characterize the ubiquitin E3 ligase MREL57 (MICROTUBULE-RELATED E3 LIGASE57) and the microtubule-stabilizing protein WDL7 (WAVE-DAMPENED2-LIKE7) in Arabidopsis and show that the MREL57-WDL7 module regulates microtubule disassembly to mediate stomatal closure in response to drought stress and ABA treatment. MREL57 interacts with, ubiquitinates and degrades WDL7, and this effect is clearly enhanced by ABA. ABA-induced stomatal closure and microtubule disassembly are significantly suppressed in mrel57 mutants, and these phenotypes can be restored when WDL7 expression is decreased. Our results unravel UPS-dependent mechanisms and the role of an MREL57-WDL7 module in microtubule disassembly and stomatal closure in response to drought stress and ABA.

An extensive plasmid library for preparing tau variants and studying their functional biochemistry

AbstractBackgroundWhile tau supports axonal transport by modulating microtubule stability, pathologically it can form neurofibrillary tangles, a key hallmark of Alzheimer’s disease and other tauopathies. Significantly, familial mutations and specific truncations regulate the biochemical processes involved in tau pathology. With the complexity and limited supply of human brain samples for research purposes, recombinant protein technology has become vital for studying tau structure and function. However, open‐source and well‐characterized plasmids and methods for expressing and purifying tau variants are lacking.MethodWe generated 58 plasmids, encoding different tau variants and verified their identities by DNA sequencing. Purification of E. coli‐expressed constructs was achieved by immobilized metal affinity chromatography followed by size exclusion chromatography. Subsequently, tau monomers were utilized to prepare oligomers and paired helical filaments (PHFs), characterized by circular dichroism (CD), transmission electron microscopy and dot blotting. Cellular uptake and cytotoxicity of stabilized oligomers were studied in neuroblastoma cells and stem cell‐derived neurons.ResultTwo sets of plasmids were generated: (i) tau fragments (14 plasmids; Figure 1), including disease‐associated fragments identified directly from human brain samples. The constructs include full‐length (FL) tau‐441 (Uniprot#P10636‐8) as well as FL tau‐441 truncated at 224, 368, 391 and 421, and their respective control fragments, and (ii) tau mutation variants (44 plasmids; Figure 2), including those encoding FL tau‐441, its four‐repeat microtubule‐binding (K18) fragment, and their respective selected familial pathological variants (N279K, V337M, P301L, C291R and S356T). Since cysteine content and locations can affect biochemical properties of tau, plasmids for expressing single (C291A), double (C291A/C322A) and triple (C291A/C322A/I260C) cysteine‐modified variants were also generated. Dot blotting with specific anti‐tau antibodies were used to verify purified fragments. Beta‐sheets in purified monomers increased with aggregation. Oligomers were granular‐shaped and reacted strongly with the anti‐oligomer antibody T22. PHFs had paired‐helical structures. Oligomer stabilization was achieved by cysteine‐labelling with fluorescent/non‐fluorescent maleimide derivatives. Fluorescent‐stabilized oligomers were internalized by neurons and co‐localized with endogenous tau.ConclusionWe anticipate that the plasmid library and the associated purification and characterization methods will facilitate future studies including microtubule‐binding assays, aggregation, cellular uptake, intra‐cellular toxicity, and extracellular release. Plasmids can be requested at: shorturl.at/ABDM3

Microtubule Dysfunction: A Common Feature of Neurodegenerative Diseases.

Neurons are particularly susceptible to microtubule (MT) defects and deregulation of the MT cytoskeleton is considered to be a common insult during the pathogenesis of neurodegenerative disorders. Evidence that dysfunctions in the MT system have a direct role in neurodegeneration comes from findings that several forms of neurodegenerative diseases are associated with changes in genes encoding tubulins, the structural units of MTs, MT-associated proteins (MAPs), or additional factors such as MT modifying enzymes which modulating tubulin post-translational modifications (PTMs) regulate MT functions and dynamics. Efforts to use MT-targeting therapeutic agents for the treatment of neurodegenerative diseases are underway. Many of these agents have provided several benefits when tested on both in vitro and in vivo neurodegenerative model systems. Currently, the most frequently addressed therapeutic interventions include drugs that modulate MT stability or that target tubulin PTMs, such as tubulin acetylation. The purpose of this review is to provide an update on the relevance of MT dysfunctions to the process of neurodegeneration and briefly discuss advances in the use of MT-targeting drugs for the treatment of neurodegenerative disorders.

Suppression of Patronin deficiency by altered Hippo signaling in Drosophila organ development

The microtubule network is crucial for cell structure and function. Patronin is a conserved protein involved in protecting the minus end of microtubules. Conversely, Klp10A is a kinesin-like microtubule depolymerase. Here we report the role of Drosophila Patronin and Klp10A for cell survival in developing organs. Loss of Patronin reduces the size of organs by activation of a caspase in imaginal discs. Reduced wing by Patronin RNAi is suppressed by knockdown of Spastin (Spas) but not Katanin 60, suggesting that Patronin is inhibitory to the severing function of Spas at the minus end. Patronin RNAi phenotype is also recovered by overexpressing Death-associated inhibitor of apoptosis 1 (Diap1), a Yorkie target gene. Heterozygote mutations in Hippo pathway genes, including hippo and warts (wts), suppress the Patronin RNAi wing phenotypes. Furthermore, Patronin physically interacts with Merlin and Expanded while reducing their function. Patronin and Klp10A antagonistically regulate their levels. Wing phenotypes of Patronin RNAi are rescued by knockdown of Klp10A, consistent with their antagonistic interaction. Klp10A overexpression also causes organ size reduction that is partially suppressed by Diap1 overexpression or wts heterozygote mutation. Taken together, this study suggests that the antagonistic interaction between Patronin and Klp10A is required for controlling cell survival and organ size by modulating microtubule stability and Hippo components.

Pharmaceutical-Grade Rigosertib Is a Microtubule-Destabilizing Agent

We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker etal., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and invitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.

Effects of salt loading on the organisation of microtubules in rat magnocellular vasopressin neurones.

Magnocellular vasopressin (VP) neurones are activated by increases in blood osmolality, leading to the secretion of VP into the circulation to promote water retention in the kidney, thus constituting a key mechanism for the regulation of body fluid homeostasis. However, chronic high salt intake can lead to excessive activation of VP neurones and increased circulating levels of VP, contributing to an elevation in blood pressure. Multiple extrinsic factors, such as synaptic inputs and glial cells, modulate the activity of VP neurones. Moreover, magnocellular neurones are intrinsically osmosensitive, and are activated by hypertonicity in the absence of neighbouring cells or synaptic contacts. Hypertonicity triggers cell shrinking, leading to the activation of VP neurones. This cell-autonomous activation is mediated by a scaffold of dense somatic microtubules, uniquely present in VP magnocellular neurones. Treating isolated magnocellular neurones with drugs modulating microtubule stability modifies the sensitivity of neuronal activation in response to acute hypertonic stimuli. However, whether the microtubule network is altered in conditions associated with enhanced neuronal activation and increased VP release, such as chronic high salt intake, remains unknown. We examined the organisation of microtubules in VP neurones of the supraoptic and paraventricular hypothalamic nuclei (SON and PVN, respectively) of rats subjected to salt-loading (drinking 2% NaCl for 7days). Using super-resolution imaging, we found that the density of microtubules in magnocellular VP neurones from the SON and PVN was significantly increased, whereas the density and organisation of microtubules remain unchanged in other hypothalamic neurones, as well as in neurones from other brain areas (e.g., hippocampus, cortex). We propose that the increase in microtubule density in magnocellular VP neurones in salt-loading promotes their enhanced activation, possibly contributing to elevated blood pressure in this condition.

MicroRNA‐181a mediates neuronal differentiation and modulates microtubule stability.

Stroke remains among the top causes of death and is the leading cause of persisting neurological disability in the United States. Treatment options remain limited to early reperfusion with thrombolysis or clot retrieval, and therapies capable of repairing damage resulting from ischemic insult are needed. Microtubules (MTs) have been recently recognized as a potential target for post‐injury therapy in the central nervous system. MTs consist of a stable and labile domain, which differ in their resistance to depolymerization, and by modulating intrinsic mechanisms that work to regulate the proportions of stable and labile MT mass in the axon, we can restore MT integrity after injury. MicroRNAs (miRNAs) are ~22‐nucleotide RNAs that regulate gene expression. MicroRNA‐181a (miR‐181a) is enriched in astrocytes, critical regulators of neuronal homeostasis and neurogenesis. Mir‐181a increases in the brain after stroke, and inhibition of miR‐181a improves functional outcomes in animal models. Using primary neuronal stem cell cultures, we demonstrate here that miR‐181a regulates neuronal differentiation. In situ analysis identified miR‐181a as a likely regulator of MT dynamics through the modulation of the MAPT (tau), MAP1B, and FIGN (fidgetin) genes. MAPT (Tau) and MAP1B are microtubule‐associated proteins (MAPs) that have been demonstrated to contribute to MT stability and neurogenesis, while fidgetin is a severing protein thought to target the labile domain of MTs and modulates a cell's ability to migrate and regulates neuronal elaboration. Therefore, we tested the hypothesis that modulation of miR‐181a activity alters MT integrity in primary neuron and astrocyte cultures and N2a cell line cultures. Immunostaining and biochemical analyses revealed injury and miR‐181a dependent changes to MAPs implicated in the modulation of microtubule stability. Overall, these results demonstrate that microRNAs can be a novel and effective target for restoring MT stability after ischemic brain injury, as well as advancing our understanding of the contributions of astrocyte regulation to increase neurogenesis and post‐injury repair after stroke.Support or Funding InformationAmerican Heart Association Fellow to Faculty Transition award: No. 14FTF19970029This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SPARC Regulates Transforming Growth Factor Beta Induced (TGFBI) Extracellular Matrix Deposition and Paclitaxel Response in Ovarian Cancer Cells.

TGFBI has been shown to sensitize ovarian cancer cells to the cytotoxic effects of paclitaxel via an integrin receptor-mediated mechanism that modulates microtubule stability. Herein, we determine that TGFBI localizes within organized fibrillar structures in mesothelial-derived ECM. We determined that suppression of SPARC expression by shRNA decreased the deposition of TGFBI in mesothelial-derived ECM, without affecting its overall protein expression or secretion. Conversely, overexpression of SPARC increased TGFBI deposition. A SPARC-YFP fusion construct expressed by the Met5a cell line co-localized with TGFBI in the cell-derived ECM. Interestingly, in vitro produced SPARC was capable of precipitating TGFBI from cell lysates dependent on an intact SPARC carboxy-terminus with in vitro binding assays verifying a direct interaction. The last 37 amino acids of SPARC were shown to be required for the TGFBI interaction while expression of a SPARC-YFP construct lacking this region (aa 1–256) did not interact and co-localize with TGFBI in the ECM. Furthermore, ovarian cancer cells have a reduced motility and decreased response to the chemotherapeutic agent paclitaxel when plated on ECM derived from mesothelial cells lacking SPARC compared to control mesothelial-derived ECM. In conclusion, SPARC regulates the fibrillar ECM deposition of TGFBI through a novel interaction, subsequently influencing cancer cell behavior.

Sustained high levels of neuroprotective, high molecular weight, phosphorylated tau in the longest-lived rodent

Tau protein is primarily expressed in neuronal axons and modulates microtubule stability. Tau phosphorylation, aggregation, and subcellular mislocalization coincide with neurodegeneration in numerous diseases, including Alzheimer's disease (AD). During AD pathogenesis, tau misprocessing accompanies Aß accumulation; however, AD animal models, despite elevated Aß, fail to develop tauopathy. To assess whether lack of tau pathology is linked to short life span common to most AD models, we examined tau processing in extraordinarily long-lived, mouse-sized naked mole-rats (NMRs; approximately 32 years), which express appreciable levels of Aß throughout life. We found that NMRs, like other mammals, display highest tau phosphorylation during brain development. Although tau phosphorylation decreases with aging, unexpectedly adult NMRs have higher levels than transgenic mice overexpressing mutant human tau. However, in sharp contrast with the somatodendritic accumulation of misprocessed tau in the transgenic mice, NMRs maintain axonal tau localization. Intriguingly, the adult NMR tau protein is 88 kDa, much larger than 45–68 kDa tau expressed in other mammals. We propose that this 88 kDa tau protein may offer exceptional microtubule stability and neuroprotection against lifelong, elevated Aß.

Microtubule dynamics in axon guidance.

Precise modulation of the cytoskeleton is involved in a variety of cellular processes including cell division, migration, polarity, and adhesion. In developing post-mitotic neurons, extracellular guidance cues not only trigger signaling cascades that act at a distance to indirectly regulate microtubule distribution, and assembly and disassembly in the growth cone, but also directly modulate microtubule stability and dynamics through coupling of guidance receptors with microtubules to control growth-cone turning. Microtubule-associated proteins including classical microtubule-associated proteins and microtubule plus-end tracking proteins are required for modulating microtubule dynamics to influence growth-cone steering. Multiple key signaling components, such as calcium, small GTPases, glycogen synthase kinase-3β, and c-Jun N-terminal kinase, link upstream signal cascades to microtubule stability and dynamics in the growth cone to control axon outgrowth and projection. Understanding the functions and regulation of microtubule dynamics in the growth cone provides new insights into the molecular mechanisms of axon guidance.

Cell Cycle Regulators Interact with Pathways That Modulate Microtubule Stability in Saccharomyces cerevisiae

The integrity of mitosis is dependent upon strict regulation of microtubule stability and dynamics. Although much information has been accumulated on regulators of the microtubule cytoskeleton, our knowledge of the specific pathways involved is still limited. Here we designed genetic screens to identify regulators of microtubule stability that are dispensable in the wild type yet become essential under microtubule-disrupting conditions. We found that the transcriptional cofactor Swi6p and activator Swi4p, as well as the G(2)/M-specific cyclin Clb2p, are required in a microtubule-destabilizing environment. Swi6p and Swi4p can combine as a transcriptional complex, called the SBF complex (SBF for Swi4/6 cell cycle box [SCB]-binding factor) that is functionally homologous to the metazoan DP1/2-E2F complex and that controls the G(1)/S transition through the genes it regulates. We show that Swi6p's contribution to microtubule stability can be either dependent or independent of the SBF complex. The SBF-dependent pathway requires downregulation of SBF complex levels and may thereby reroute the transcriptional program in favor of greater microtubule stability. This pathway can be triggered by overexpression of Fcp1p, a phosphatase in the general transcription machinery, or by expression of an allele of SWI6 that is associated with reduced transcription from SBF-controlled promoters. The SBF-independent pathway is activated by a constitutively nuclear allele of Swi6p. Our results introduce novel roles in microtubule stability for genes whose participation in the process may be masked under normal conditions yet nonetheless acquire a dominant role when microtubule stability is compromised.

Abstract 2539: Reactivating tumor suppression with small-molecule deregulators of the Rho-regulated diaphanous-related formin autoinhibitory mechanism

Abstract Loss of all or part of the long-arm of chromosome 5 is a frequent karyotypic defect in myeloproliferitive neoplasms, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). To date, there is no clear mechanism that accounts for how any of the 5q tumor suppressor candidates contribute to hematopoietic stem cell growth control or how defects in their expression/function contribute to malignant progression. An attractive 5q- MDS tumor suppressor gene is DIAPH1 (5q31.3)]. DIAPH1 encodes the canonical mammalian Diaphanous-related (mDia) formin mDia1. Consistent with a role in tumor suppression, mDia1 knockout (KO) mice develop an age-dependent MDS-like phenotype. mDia1 is an effector for the Rho-family small GTP-binding protein RhoB, which is known to harbor tumor suppressor activity. mDia1 has a central role in the actin and microtubule dynamics that underlie cell division and directed migration. In this study we sought to determine if we could manipulate mDia activity to ‘reactivate’ tumor suppression by exploiting our working knowledge of the Rho-controlled mDia autoregulatory mechanism. GTP-bound Rho proteins activate mDia formins by binding to and disrupting an intramolecular autoinhibitory mechanism mediated by regulatory Dia-inhibitory (DID) and Dia-autoregulatory (DAD) domains that flank the conserved formin homology-2 (FH2) domain found in all formins that is responsible for assembling linear actin filaments and modulating microtubule stability. To identify small molecule activators of mDia formins, a screen for inhibitors of DID-DAD binding was conducted in a high-throughput format. We identified two structurally-related semi-thiocarbazones that bind within the DAD binding pocket of DID. These compounds, called Intramimics (IMM)-1 and −2, possess IC50 values for in vitro inhibition of DAD binding of 99 and 140 nM, respectively; the values were comparable to a DAD-derived peptide (280 nM). Consistent with mDia activation, exposure of fibroblasts and AML-derived cells leads to stabilization of actin and microtubule dynamics. The consequence is the induction of apoptosis, which was assayed by a variety of approaches. Using IMMs as tool compounds to explore tumor suppression mechanisms, IMM treatment activated serum response factor (SRF)-regulated gene expression. SRF is a growth factor-regulated transcription factor that also senses changes in actin assembly and is a potent activator of Egr1 expression. Egr1 is another 5q tumor suppressor candidate and is also a transcription factor that appears to be a master regulator of the well-known tumor suppressors PTEN and p53. Our next goal is to test IMMs in preclinical models for their ability to modulate myeloproliferation and to inhibit tumor growth, alone and in combination with other anti-tumor agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2539.

Leucine‐rich repeat kinase 2 phosphorylates brain tubulin‐beta isoforms and modulates microtubule stability – a point of convergence in Parkinsonian neurodegeneration?

Autosomal dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset Parkinson's disease. The most prevalent LRRK2(G2019S) mutation has repeatedly been shown to enhance kinase activity and neurotoxicity, however, the molecular mechanisms leading to neurodegeneration remain poorly defined. Here we show that recombinant human LRRK2 preferentially phosphorylates tubulin-beta purified from bovine brain and that phosphorylation is three-fold enhanced by the LRRK2(G2019S) mutation. By tandem mass spectrometry, Thr107 was identified as phosphorylation site which is highly conserved between tubulin-beta family members and also between tubulin-beta genes of different species. LRRK2 was co-immunoprecipitated with tubulin-beta both from wild-type mouse brain and from LRRK2 over-expressing, non-neuronal human embryonic kidney 293 cells. However, an effect of LRRK2 on tubulin phosphorylation and assembly was only detectable in mouse brain samples. In vitro co-incubation of bovine brain tubulins with LRRK2 increased microtubule stability in the presence of microtubule-associated proteins which may explain the reduction in neurite length in LRRK2-deficient neurons in culture. These findings suggest that LRRK2(G2019S)-induced neurodegeneration in Parkinsonian brains may be partly mediated by increased phosphorylation of tubulin-beta and constraining of microtubule dynamics.

Serine Protease HtrA1 Associates with Microtubules and Inhibits Cell Migration

HtrA1 belongs to a family of serine proteases found in organisms ranging from bacteria to humans. Bacterial HtrA1 (DegP) is a heat shock-induced protein that behaves as a chaperone at low temperature and as a protease at high temperature to help remove unfolded proteins during heat shock. In contrast to bacterial HtrA1, little is known about the function of human HtrA1. Here, we report the first evidence that human HtrA1 is a microtubule-associated protein and modulates microtubule stability and cell motility. Intracellular HtrA1 is localized to microtubules in a PDZ (PSD95, Dlg, ZO1) domain-dependent, nocodazole-sensitive manner. During microtubule assembly, intracellular HtrA associates with centrosomes and newly polymerized microtubules. In vitro, purified HtrA1 promotes microtubule assembly. Moreover, HtrA1 cosediments and copurifies with microtubules. Purified HtrA1 associates with purified alpha- and beta-tubulins, and immunoprecipitation of endogenous HtrA1 results in coprecipitation of alpha-, beta-, and gamma-tubulins. Finally, downregulation of HtrA1 promotes cell motility, whereas enhanced expression of HtrA1 attenuates cell motility. These results offer an original identification of HtrA1 as a microtubule-associated protein and provide initial mechanistic insights into the role of HtrA1 in the regulation of cell motility by modulating microtubule stability.

PASK (proline–alanine-rich Ste20-related kinase) binds to tubulin and microtubules and is involved in microtubule stabilization

Proline–alanine-rich Ste20-related kinase (PASK, also referred to as SPAK) has been linked to ion transport regulation. Here, we report two novel activities of PASK: binding to tubulin and microtubules and the promotion of microtubule assembly. Tubulin binding assay showed that full-length PASK and its kinase domain bound to purified tubulin whereas the N-terminal or C-terminal non-catalytic domains of PASK did not. The full-length PASK and its kinase domain were sedimented with paclitaxel-stabilized microtubules by ultracentrifugation. These results indicate that the kinase domain of PASK can interact directly with both microtubules and soluble tubulin in vitro. Truncated PASK lacking the N-terminal non-catalytic domain promoted microtubule assembly at a subcritical concentration of purified tubulin. FLAG–PASK expressed in COS-7 cells translocated to the cytoskeleton when the cells were stimulated with hypertonic sodium chloride, and stabilized microtubules against depolymerization by nocodazole. Our findings suggest that PASK may regulate the cytoskeleton by modulating microtubule stability.

Septins: New Microtubule Interacting Partners

Originally characterized as regulators of cytokinesis, septins were later implicated in other cellular processes. Recent studies show that septins have a broader role in microtubule-dependent processes, such as karyokinesis, exocytosis, and maintenance of cell shape. Many members of the septin family have been shown to colocalize or interact with the microtubule cytoskeleton, suggesting that these might be general properties of septins. Septins could play an important role in regulating microtubule dynamics by interacting with microtubule-associated proteins (MAPs) that modulate microtubule stability. Being able to associate with both microtubules and actin, septins can play an important role as adaptors between the two cytoskeletons and as regulators of processes in which both actin and microtubules are involved. As septins are associated with various neurodegenerative diseases and cancer, a better understanding of the biology of septins and their interactions with microtubules is important in order to develop possible therapeutic strategies for these diseases.

The Extracellular Matrix Protein TGFBI Induces Microtubule Stabilization and Sensitizes Ovarian Cancers to Paclitaxel

SummaryThe extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability.

Tobacco mosaic virus movement protein functions as a structural microtubule-associated protein.

The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.