Determination Of Cell Polarity Research Articles

Structural analysis of microtubule binding by minus-end targeting protein Spiral2

Microtubules (MTs) are dynamic cytoskeletal polymers that play a critical role in determining cell polarity and shape. In plant cells, acentrosomal MTs are localized on the cell surface and are referred to as cortical MTs. Cortical MTs nucleate in the cell cortex and detach from nucleation sites. The released MT filaments perform treadmilling, with the plus-ends of MTs polymerizing and the minus-ends depolymerizing. Minus-end targeting proteins, -TIPs, include Spiral2, which regulates the minus-end dynamics of acentrosomal MTs. Spiral2 accumulates autonomously at MT minus-ends and inhibits filament shrinkage, but the mechanism by which Spiral2 specifically recognizes minus-ends of MTs remains unknown. Here we describe the crystal structure of Spiral2's N-terminal MT-binding domain. The structural properties of this domain resemble those of the HEAT repeat structure of the tumor overexpressed gene (TOG) domain, but the number of HEAT repeats is different and the conformation is highly arched. Gel filtration and co-sedimentation analyses demonstrate that the domain binds preferentially to MT filaments rather than the tubulin dimer, and that the tubulin-binding mode of Spiral2 via the basic surface is similar to that of the TOG domain. We constructed an in silico model of the Spiral2-tubulin complex to identify residues that potentially recognize tubulin. Mutational analysis revealed that the key residues inferred in the model are involved in microtubule recognition, and provide insight into the mechanism by which end-targeting proteins stabilize MT ends.

HDA6 modulates Arabidopsis pavement cell morphogenesis through epigenetic suppression of ROP6 GTPase expression and signaling.

The transcriptional regulation of Rho-related GTPase from plants (ROPs), which determine cell polarity formation and maintenance during plant development, still remains enigmatic. In this study, we elucidated the epigenetic mechanism of histone deacetylase HDA6 in transcriptional repression of ROP6 and its impact on cell polarity and morphogenesis in Arabidopsis leaf epidermal pavement cells (PCs). We found that the hda6 mutant axe1-4 exhibited impaired jigsaw-shaped PCs and convoluted leaves. This correlated with disruptions in the spatial organizations of cortical microtubules and filamentous actin, which is integral to PC indentation and lobe formation. Further transcriptional analyses and chromatin immunoprecipitation assay revealed that HDA6 specifically represses ROP6 expression through histone H3K9K14 deacetylation. Importantly, overexpression of dominant negative-rop6 in axe1-4 restored interdigitated cell morphology. Our study unveils HDA6 as a key regulator in Arabidopsis PC morphogenesis through epigenetic suppression of ROP6. It reveals the pivotal role of HDA6 in the transcriptional regulation of ROP6 and provides compelling evidence for the functional interplay between histone deacetylation and ROP6-mediated cytoskeletal arrangement in the development of interdigitated PCs.

Regulation of RNA localization during oocyte maturation by dynamic RNA-ER association and remodeling of the ER

Asymmetric localization of mRNAs is crucial for cell polarity and cell fate determination. By performing fractionation RNA-seq, we report here that a large number of maternal RNAs are associated with the ER in Xenopus oocytes but are released into the cytosol after oocyte maturation. We provide evidence that the majority of ER-associated RNA-binding proteins (RBPs) remain associated with the ER after oocyte maturation. However, all ER-associated RBPs analyzed exhibit reduced binding to some of their target RNAs after oocyte maturation. Our results further show that the ER is remodeled massively during oocyte maturation, leading to the formation of a widespread tubular ER network in the animal hemisphere that is required for the asymmetric localization of mRNAs in mature eggs. Thus, our findings demonstrate that dynamic regulation of RNA-ER association and remodeling of the ER are important for the asymmetric localization of RNAs during development.

The JMJD6/HURP axis promotes cell migration via NF-κB-dependent centrosome repositioning and Cdc42-mediated Golgi repositioning.

Golgi apparatus (GA) and centrosome reposition toward cell leading end during directional cell migration in a coupling way, thereby determining cell polarity by transporting essential factors to the proximal plasma membrane. The study provides mechanistic insights into how GA repositioning (GR) is regulated, and how GR and centrosome repositioning (CR) are coupled. Our previous published works reveals that PRMT5 methylates HURP at R122 and the HURP m122 inhibits GR and cell migration by stabilizing GA-associated acetyl-tubulin and then rigidifying GA. The current study further shows that the demethylase JMJD6-guided demethylation of HURP at R122 promotes GR and cell migration. The HURP methylation mimicking mutant 122 F blocks JMJD6-induced GR and cell migration, suggesting JMJD6 relays GR stimulating signal to HURP. Mechanistic studies reveal that the HURP methylation deficiency mutant 122 K promotes GR through NF-κB-induced CR and subsequently CR-dependent Cdc42 upregulation, where Cdc42 couples CR to GR. Taken together, HURP methylation statuses provide a unique opportunity to understand how GR is regulated, and the GA intrinsic mechanism controlling Golgi rigidity and the GA extrinsic mechanism involving NF-κB-CR-Cdc42 cascade collectively dictate GR.

Aberrant activation of p53/p66Shc-mInsc axis increases asymmetric divisions and attenuates proliferation of aged mammary stem cells.

Aging is accompanied by the progressive decline in tissue regenerative capacity and functions of resident stem cells (SCs). Underlying mechanisms, however, remain unclear. Here we show that, during chronological aging, self-renewing mitoses of mammary SCs (MaSCs) are preferentially asymmetric and that their progeny divides less frequently, leading to decreased number of MaSCs and reduced regenerative potential. Underlying mechanisms are investigated in the p66Shc-/- mouse, which exhibits several features of delayed aging, including reduced involution of the mammary gland (MG). p66Shc is a mitochondrial redox sensor that activates a specific p53 transcriptional program, in which the aging-associated p44 isoform of p53 plays a pivotal role. We report here that aged p66Shc-/- MaSCs show increased symmetric divisions, increased proliferation and increased regenerative potential, to an extent reminiscent of young wild-type (WT) MaSCs. Mechanistically, we demonstrate that p66Shc, together with p53: (i) accumulates in the aged MG, (ii) sustains expression of the cellpolarity determinant mInscuteable and, concomitantly, (iii) down-regulates critical cellcycle genes (e.g.,: Cdk1 and Cyclin A). Accordingly, overexpression of p53/p44 increases asymmetric divisions and decreases proliferation of young WT MaSCs in a p66Shc-dependent manner and overexpression of mInsc restores WT-like levels of asymmetric divisions in aged p66Shc-/- MaSCs. Notably, deletion of p66Shc has negligible effects in young MaSCs and MG development. These results demonstrate that MG aging is due to aberrant activation of p66Shc, which induces p53/p44 signaling, leading to failure of symmetric divisions, decreased proliferation and reduced regenerative potential of MaSCs.

Membrane phosphoinositides and renal epithelial cell polarity determination in the Xenopus pronephros in vivo

BackgroundThough they are only minor components of cell membranes, phosphoinositide lipids (PIs) participate in numerous signaling processes and in membrane identity determination. Many studies of cultured MDCK renal epithelial cells found that PIs are distributed with polarity among plasma membrane (PM) domains, and that their polarized distributions are required for the delivery of distinct populations of apical and basolateral membrane proteins. The extent to which PIs drive these processes in actual renal epithelial cells in vivo has never been examined. We investigate the distribution of PIs in vivo in the pronephros of Xenopus laevis tadpoles using MCherry‐tagged Pleckstrin Homology (PH) domains that selectively bind different PIs (PH‐AKT, PH‐PLCD1, which bind to (PI(3,4,5,)P3 and PI(4,5)P2 respectively) with the goal of assessing whether and how PI localizations affect cell polarity determination and the trafficking of proteins to their sites of ultimate functional residence in renal epithelial cells in situ.MethodsmRNA encoding MCherry‐PH‐AKT or MCherry‐PH‐PLCD1 was injected into fertilized oocytes and their distributions were assessed in the developed pronephros at stage NF45 via fluorescence microscopy. Knockdown (KD) of PTEN, a lipid 3’ phosphatase that regulates membrane PI composition, was achieved via injection of targeted morpholinos and confirmed by western blotting. The effects of PTEN KD on MCherry‐PH‐AKT distribution were assessed by fluorescence microscopy.ResultsIn MDCK cells PH‐AKT and PH‐PLCD1 localize to the basolateral and apical PMs, respectively. Their distributions are quite different in the pronephros, with both sensors localizing apically in the proximal portion of the tubule and basolaterally in the distal part. Interestingly, PTEN localization, at least in proximal tubules, is apical and not different than previously reported in cell models. Finally, PH‐AKT staining dramatically re‐distributes to the lateral domain of renal cells upon PTEN KD but this treatment does not alter the localization of protein markers of epithelial PM polarity.ConclusionsThese studies constitute the first effort to assess the role of PIs in establishing PM polarity in renal epithelial cells in vivo. Our data reveal important differences from observations previously made with in vitro systems. These findings highlight the need to explore the processes that produce renal epithelial cell polarity in vivo and in the context of intact renal tubules.

The cell polarity determinant Dlg1 facilitates epithelial invagination by promoting tissue-scale mechanical coordination.

Epithelial folding mediated by apical constriction serves as a fundamental mechanism to convert flat epithelial sheets into multilayered structures. It remains unknown whether additional mechanical inputs are required for apical constriction-mediated folding. Using Drosophila mesoderm invagination as a model, we identified an important role for the non-constricting, lateral mesodermal cells adjacent to the constriction domain ('flanking cells') in facilitating epithelial folding. We found that depletion of the basolateral determinant Dlg1 disrupts the transition between apical constriction and invagination without affecting the rate of apical constriction. Strikingly, the observed delay in invagination is associated with ineffective apical myosin contractions in the flanking cells that lead to overstretching of their apical domain. The defects in the flanking cells impede ventral-directed movement of the lateral ectoderm, suggesting reduced mechanical coupling between tissues. Specifically disrupting the flanking cells in wild-type embryos by laser ablation or optogenetic depletion of cortical actin is sufficient to delay the apical constriction-to-invagination transition. Our findings indicate that effective mesoderm invagination requires intact flanking cells and suggest a role for tissue-scale mechanical coupling during epithelial folding.

Ultrastructure of the foliose lichen Myelochroa leucotyliza and its solo fungal and algal (Trebouxia sp.) partners

Lichens are slow-growing niche-constructing organisms formed via scripted symbiotic/mutualist relationships between fungi, algae, and bacteria. We have used quick-freeze deep-etch electron microscopy (QFDEEM), coupled with several additional microscopic techniques, to analyze the Japanese foliose lichen Myelochroa leucotyliza. We have also examined its constituent fungi and algae grown separately (solo) and in co-culture in the laboratory, as well as solo fungi incubated with conditioned algal media (CM) for 9 days. We report our general findings on M. leucotyliza architecture in a Supplement, and focus this report on three findings of particular interest. 1) Plasma membrane differentiations called nubbins are found adjacent to, and at the center of, the cell-wall septa in both lichenized and solo hyphae, suggesting a role in cell polarity determination and/or cell-cell communication within the hyphae. 2) Nubbins also localize to the tips of hyphal sub-branches produced by the lichenized but not the solo fungi. These in turn form tip-to-tip contacts (bridges) with one another, suggesting a role in inter-hyphal communication. 3) CM treatment of solo fungi induces a) the production of secondary metabolites, polysaccharides, and lipids; b) a transformation of ER topology and its association with the plasma membrane; and c) a dramatic change in vacuole organization, indicating that the algal growth medium contains soluble materials that trigger fungal differentiation.

Protein phosphatase SppA regulates apical growth and dephosphorylates cell polarity determinant DivIVA in Streptomyces coelicolor.

Members of the Actinobacteria, including mycobacteria and streptomycetes, exhibit a distinctive mode of polar growth, with cell wall synthesis occurring in zones at cell poles and directed by the essential cell polarity determinant DivIVA. Streptomyces coelicolor modulates polar growth via the Ser/Thr protein kinase AfsK, which phosphorylates DivIVA. Here, we show that the phosphoprotein phosphatase SppA has strong effects on polar growth and cell shape and that it reverses the AfsK-mediated phosphorylation of DivIVA. SppA affects hyphal branching and the rate of tip extension. The sppA mutant hyphae also exhibit a high frequency of spontaneous growth arrests, indicating problems with maintenance of tip extension. The phenotypic effects are partially suppressed in an afsK sppA double mutant, indicating that AfsK and SppA to some extent share target proteins. Strains with a nonphosphorylatable mutant DivIVA confirm that the effect of afsK on hyphal branching during normal growth is mediated by DivIVA phosphorylation. However, the phenotypic effects of sppA deletion are independent of DivIVA phosphorylation and must be mediated via other substrates. This study adds a PPP-family protein phosphatase to the proteins involved in the control of polar growth and cell shape determination in S. coelicolor.

Membrane Phosphoinositides and Renal Epithelial Cell Polarity Determination in the Xenopus Pronephros In Vivo

Membrane Phosphoinositides and Renal Epithelial Cell Polarity Determination in the Xenopus Pronephros In Vivo

The PRMT5/HURP axis retards Golgi repositioning by stabilizing acetyl‐tubulin and Golgi apparatus during cell migration

The Golgi apparatus (GA) translocates to the cell leading end during directional migration, thereby determining cell polarity and transporting essential factors to the migration apparatus. The study provides mechanistic insights into how GA repositioning (GR) is regulated. We show that the methyltransferase PRMT5 methylates the microtubule regulator HURP at R122. The HURP methylation mimicking mutant 122F impairs GR and cell migration. Mechanistic studies revealed that HURP 122F or endogenous methylated HURP, that is, HURP m122, interacts with acetyl-tubulin. Overexpression of HURP 122F stabilizes the bundling pattern of acetyl-tubulin by decreasing the sensitivity of the latter to a microtubule disrupting agent nocodazole. HURP 122F also rigidifies GA via desensitizing the organelle to several GA disrupting chemicals. Similarly, the acetyl-tubulin mimicking mutant 40Q or tubulin acetyltransferase αTAT1 can rigidify GA, impair GR, and retard cell migration. Reversal of HURP 122F-induced GA rigidification, by knocking down GA assembly factors such as GRASP65 or GM130, attenuates 122F-triggered GR and cell migration. Remarkably, PRMT5 is found downregulated and the level of HURP m122 is decreased during the early hours of wound healing-based cell migration, collectively implying that the PRMT5-HURP-acetyl-tubulin axis plays the role of brake, preventing GR and cell migration before cells reach empty space.

Discovery of a small protein-encoding cis-regulatory overlapping gene of the tumor suppressor gene Scribble in humans

Intensive gene annotation has revealed many functional and regulatory elements in the human genome. Although eukaryotic protein-coding genes are generally transcribed into monocistronic mRNAs, recent studies have discovered additional short open reading frames (sORFs) in mRNAs. Here, we performed proteogenomic data mining for hidden proteins categorized into sORF-encoded polypeptides (SEPs) in human cancers. We identified a new SEP-encoding overlapping sORF (oORF) on the cell polarity determinant Scribble (SCRIB) that is considered a proto-oncogene with tumor suppressor function in Hippo-YAP/TAZ, MAPK/ERK, and PI3K/Akt/mTOR signaling. Reanalysis of clinical human proteomic data revealed translational dysregulation of both SCRIB and its oORF, oSCRIB, during carcinogenesis. Biochemical analyses suggested that the translatable oSCRIB constitutively limits the capacity of eukaryotic ribosomes to translate the downstream SCRIB. These findings provide a new example of cis-regulatory oORFs that function as a ribosomal roadblock and potentially serve as a fail-safe mechanism to normal cells for non-excessive downstream gene expression, which is hijacked in cancer.

Regulation of Fibroblast Cell Polarity by Src Tyrosine Kinase.

Src protein tyrosine kinases (SFKs) are a family of nonreceptor tyrosine kinases that are localized beneath the plasma membrane and are activated during cell adhesion, migration, and elongation. Due to their involvement in the activation of signal transduction cascades, SFKs have been suggested to play important roles in the determination of cell polarity during cell extension and elongation. However, the mechanism underlying Src-mediated polarity formation remains unclear. The present study was performed to investigate the mechanisms underlying Src-induced cell polarity formation and cell elongation using Src knockout fibroblasts (SYFs) together with an inhibitor of Src. Normal and Src knockout fibroblasts were also transfected with a wild-type c-Src, dominant negative c-Src, or constitutively active c-Src gene to analyze the changes in cell morphology. SYF cells cultured on a glass substrate elongated symmetrically into spindle-shaped cells, with the formation of focal adhesions at both ends of the cells. When normal fibroblasts were treated with Src Inhibitor No. 5, a selective inhibitor of Src tyrosine kinases, they elongated into symmetrical spindle-shaped cells, similar to SYF cells. These results suggest that cell polarity during extension and elongation may be regulated by SFKs and that the expression and regulation of Src are important for the formation of polarity during cell elongation.

Puromycin-sensitive aminopeptidase is required for C2C12 myoblast proliferation and differentiation.

The ubiquitin‐proteasome system is a major protein degradation pathway in the cell. Proteasomes produce several peptides that are rapidly degraded to free amino acids by intracellular aminopeptidases. Our previous studies reported that proteolysis via proteasomes and aminopeptidases is required for myoblast proliferation and differentiation. However, the role of intracellular aminopeptidases in myoblast proliferation and differentiation had not been clarified. In this study, we investigated the effects of puromycin‐sensitive aminopeptidase (PSA) on C2C12 myoblast proliferation and differentiation by knocking down PSA. Aminopeptidase enzymatic activity was reduced in PSA‐knockdown myoblasts. Knockdown of PSA induced impaired cell cycle progression in C2C12 myoblasts and accumulation of cells at the G2/M phase. Additionally, after the induction of myogenic differentiation in PSA‐knockdown myoblasts, multinucleated circular‐shaped myotubes with impaired cell polarity were frequently identified. Cell division cycle 42 (CDC42) knockdown in myoblasts resulted in a loss of cell polarity and the formation of multinucleated circular‐shaped myotubes, which were similar to PSA‐knockdown myoblasts. These data suggest that PSA is required for the proliferation of myoblasts in the growth phase and for the determination of cell polarity and elongation of myotubes in the differentiation phase.

The formation of paranodal spirals at the ends of CNS myelin sheaths requires the planar polarity protein Vangl2.

During axonal ensheathment, noncompact myelin channels formed at lateral edges of the myelinating process become arranged into tight paranodal spirals that resemble loops when cut in cross section. These adhere to the axon, concentrating voltage-dependent sodium channels at nodes of Ranvier and patterning the surrounding axon into distinct molecular domains. The signals responsible for forming and maintaining the complex structure of paranodal myelin are poorly understood. Here, we test the hypothesis that the planar cell polarity determinant Vangl2 organizes paranodal myelin. We show that Vangl2 is concentrated at paranodes and that, following conditional knockout of Vangl2 in oligodendrocytes, the paranodal spiral loosens, accompanied by disruption to the microtubule cytoskeleton and mislocalization of autotypic adhesion molecules between loops within the spiral. Adhesion of the spiral to the axon is unaffected. This results in disruptions to axonal patterning at nodes of Ranvier, paranodal axon diameter and conduction velocity. When taken together with our previous work showing that loss of the apico-basal polarity protein Scribble has the opposite phenotype-loss of axonal adhesion but no effect on loop-loop autotypic adhesion-our results identify a novel mechanism by which polarity proteins control the shape of nodes of Ranvier and regulate conduction in the CNS.

The cytoskeletal motor proteins Dynein and MyoV direct apical transport of Crumbs

Crumbs (Crb in Drosophila; CRB1-3 in mammals) is a transmembrane determinant of epithelial cell polarity and a regulator of Hippo signalling. Crb is normally localized to apical cell-cell contacts, just above adherens junctions, but how apical trafficking of Crb is regulated in epithelial cells remains unclear. We use the Drosophila follicular epithelium to demonstrate that polarized trafficking of Crb is mediated by transport along microtubules by the motor protein Dynein and along actin filaments by the motor protein Myosin-V (MyoV). Blocking transport of Crb-containing vesicles by Dynein or MyoV leads to accumulation of Crb within Rab11 endosomes, rather than apical delivery. The final steps of Crb delivery and stabilisation at the plasma membrane requires the exocyst complex and three apical FERM domain proteins – Merlin, Moesin and Expanded – whose simultaneous loss disrupts apical localization of Crb. Accordingly, a knock-in deletion of the Crb FERM-binding motif (FBM) also impairs apical localization. Finally, overexpression of Crb challenges this system, creating a sensitized background to identify components involved in cytoskeletal polarization, apical membrane trafficking and stabilisation of Crb at the apical domain.

Sphingolipid biosynthetic pathway is crucial for growth, biofilm formation and membrane integrity of Scedosporium boydii.

Aim: Glycosphingolipids are conserved lipids displaying a variety of functions in fungal cells, such as determination of cell polarity and virulence. They have been considered as potent targets for new antifungal drugs. The present work aimed to test two inhibitors, myriocin and DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol, in Scedosporium boydii, a pathogenic fungus which causes a wide range of disease. Materials & methods: Mass spectrometry, microscopy and cell biology approaches showed that treatment with both inhibitors led to defects in fungal growth and membrane integrity, and caused an increased susceptibility to the current antifungal agents. Conclusion: These data demonstrate the antifungal potential of drugs inhibiting sphingolipid biosynthesis, as well as the usefulness of sphingolipids as promising targets for the development of new therapeutic options.

Notch-Mediated Determination of Hair-Bundle Polarity in Mechanosensory Hair Cells of the Zebrafish Lateral Line.

The development of mechanosensory epithelia, such as those of the auditory and vestibular systems, results in the precise orientation of mechanosensory hair cells. After division of a precursor cell in the zebrafish's lateral line, the daughter hair cells differentiate with opposite mechanical sensitivity. Through a combination of theoretical and experimental approaches, we show that Notch1a-mediated lateral inhibition produces a bistable switch that reliably gives rise to cell pairs of opposite polarity. Using a mathematical model of the process, we predict the outcome of several genetic and chemical alterations to the system, which we then confirm experimentally. We show that Notch1a downregulates the expression of Emx2, a transcription factor known to be involved in polarity specification, and acts in parallel with the planar-cell-polarity system to determine the orientation of hair bundles. By analyzing the effect of simultaneous genetic perturbations to Notch1a and Emx2, we infer that the gene-regulatory network determining cell polarity includes an undiscovered polarity effector.

A CRISPR Screen Identifies the Cell Polarity Determinant Crumbs 3 as an Adeno-associated Virus Restriction Factor in Hepatocytes.

Adeno-associated viruses (AAV) are helper-dependent parvoviruses that have been developed into promising gene therapy vectors. Many studies, including a recent unbiased genomic screen, have identified host factors essential for AAV cell entry, but no genome-wide screens that address inhibitory host factors have been reported. Here, we utilize a novel CRISPR screen to identify AAV restriction factors in a human hepatocyte cell line. The major hit from our gain-of-function screen is the apical polarity determinant Crumbs 3 (Crb3). Knockout (KO) of Crb3 enhances AAV transduction, while overexpression exerts the opposite effect. Further, Crb3 appears to restrict AAV transduction in a serotype- and cell type-specific manner. Particularly, for AAV serotype 9 and a rationally engineered AAV variant, we demonstrate that increased availability of galactosylated glycans on the surfaces of Crb3 KO cells, but not the universal AAV receptor, leads to increased capsid attachment and enhanced transduction. We postulate that Crb3 could serve as a key molecular determinant that restricts the availability of AAV glycan attachment factors on the cell surface by maintaining apical-basal polarity and tight junction integrity.IMPORTANCE Adeno-associated viruses (AAVs) have recently emerged at the forefront as gene therapy vectors; however, our understanding of host factors that influence AAV transduction in different cell types is still evolving. In the present study, we perform a genome-scale CRISPR knockout screen to identify cellular host factors that restrict AAV infection in hepatocyte cultures. We discover that Crumbs 3, which determines cellular polarity, also influences the distribution of certain carbohydrate attachment factors on the cell surface. This in turn affects the ability of virions to bind and enter the cells. This study underscores the importance of cell polarity in AAV transduction and provides a potential molecular basis for the differential infectious mechanism(s) in cell culture versus organ systems.

Neuronal microtubules and proteins linked to Parkinson's disease: a relevant interaction?

Neuronal microtubules are key determinants of cell morphology, differentiation, migration and polarity, and contribute to intracellular trafficking along axons and dendrites. Microtubules are strictly regulated and alterations in their dynamics can lead to catastrophic effects in the neuron. Indeed, the importance of the microtubule cytoskeleton in many human diseases is emerging. Remarkably, a growing body of evidence indicates that microtubule defects could be linked to Parkinson's disease pathogenesis. Only a few of the causes of the progressive neuronal loss underlying this disorder have been identified. They include gene mutations and toxin exposure, but the trigger leading to neurodegeneration is still unknown. In this scenario, the evidence showing that mutated proteins in Parkinson's disease are involved in the regulation of the microtubule cytoskeleton is intriguing. Here, we focus on α-Synuclein, Parkin and Leucine-rich repeat kinase 2 (LRRK2), the three main proteins linked to the familial forms of the disease. The aim is to dissect their interaction with tubulin and microtubules in both physiological and pathological conditions, in which these proteins are overexpressed, mutated or absent. We highlight the relevance of such an interaction and suggest that these proteins could trigger neurodegeneration via defective regulation of the microtubule cytoskeleton.