The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes

Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)‐associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR‐induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate‐treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in‐gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3ε, WASP, and WIPF1) and identified several novel putative Nck‐binding proteins. We subsequently verified the SH2 domain binding to the actin‐binding protein HIP55 and to FYB/ADAP, and the SH3‐mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR‐to‐cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes.     

Microtubule Associated Protein 1b (MAP1B) Is a Marker of the Microtubular Cytoskeleton in Podocytes but Is Not Essential for the Function of the Kidney Filtration Barrier in Mice

Podocytes are essential for the function of the kidney glomerular filter. A highly differentiated cytoskeleton is requisite for their integrity. Although much knowledge has been gained on the organization of cortical actin networks in podocyte’s foot processes, less is known about the molecular organization of the microtubular cytoskeleton in primary processes and the cell body. To gain an insight into the organization of the microtubular cytoskeleton of the podocyte, we systematically analyzed the expression of microtubule associated proteins (Maps), a family of microtubules interacting proteins with known functions as regulator, scaffold and guidance proteins. We identified microtubule associated protein 1b (MAP1B) to be specifically enriched in podocytes in human and rodent kidney. Using immunogold labeling in electron microscopy, we were able to demonstrate an enrichment of MAP1B in primary processes. A similar association of MAP1B with the microtubule cytoskeleton was detected in cultured podocytes. Subcellular distribution of MAP1B HC and LC1 was analyzed using a double fluorescent reporter MAP1B fusion protein. Subsequently we analyzed mice constitutively depleted of MAP1B. Interestingly, MAP1B KO was not associated with any functional or structural alterations pointing towards a redundancy of MAP proteins in podocytes. In summary, we established MAP1B as a specific marker protein of the podocyte microtubular cytoskeleton.     

The effect of colchicine on pyrin and pyrin interacting proteins

MEFV which encodes pyrin, cause familial Mediterranean fever (FMF), the most common auto‐inflammatory disease. Pyrin is believed to be a regulator of inflammation, though the nature of this regulatory activity remains to be identified. Prophylactic treatment with colchicine, a microtubule toxin, has had a remarkable effect on disease progression and outcome. It has been thought that, inhibition of microtubule polymerization is the main mechanism of action of colchicine. But, the exact cellular mechanism explaining the efficacy of colchicine in suppressing FMF attacks is still unclear. Given the ability of colchicine treatment to be considered as a differential diagnosis criteria of FMF, we hypothesized that colchicine may have a specific effect on pyrin and pyrin interacting proteins. This study showed that colchicine prevents reticulated fibrils formed by PSTPIP1 filaments and reduces ASC speck rates in transfected cells. We further noted that, colchicine down‐regulates MEFV expression in THP‐1 cells. We also observed that colchicine causes re‐organization of actin cytoskeleton in THP‐1 cells. Pyrin is an actin‐binding protein that specifically localizes with polymerizing actin filaments. Thus, MEFV expression might be affected by re‐organization of actin cytoskeleton. The data presented here reveal an important connection between colchicine and pyrin which might explain the remarkable efficacy of colchicine in preventing FMF attacks. J. Cell. Biochem. 113: 3536–3546, 2012. © 2012 Wiley Periodicals, Inc.     

The degradation of kinesin-like calmodulin binding protein of D. salina (DsKCBP) is mediated by the ubiquitin–proteasome system

Kinesin-like calmodulin binding protein (KCBP) is a member of kinesin-14 subfamily with unconventional domains distinct from other kinesins. This unique kinesin has the myosin tail homology 4 domain (MyTH4) and band4.1, ezrin, radixin and moesin domain (FERM) at the N-terminal which interact with several cytoskeleton proteins. Although KCBP is implicated in several microtubule-related cellular processes, studies on the KCBP of Dunaliella salina (DsKCBP) have not been reported. In this study, the roles of DsKCBP in flagella and cytoskeleton were investigated and the results showed that DsKCBP was present in flagella and upregulated during flagellar assembly indicting that it may be a flagellar kinesin and plays a role in flagellar assembly. A MyTH4-FERM domain of the DsKCBP was identified as a microtubule and actin interacting site. The interaction of DsKCBP with both microtubules and actin microfilaments suggests that this kinesin may be employed to coordinate these two cytoskeleton elements in algal cells. To gain more insights into the cellular function of the kinesin, DsKCBP-interacting proteins were examined using yeast two-hybrid screen. A 26S proteasome subunit Rpn8 was identified as a novel interacting partner of DsKCBP and the MyTH4-FERM domain was necessary for the interaction of DsKCBP with Rpn8. Furthermore, the DsKCBP was polyubiquitinated and up-regulated by proteasome inhibitor and degraded by ubiquitin–proteasome system indicating that proteasome is related to kinesin degradation.     

Riboproteomics of the hepatitis C virus internal ribosomal entry site

Hepatitis C virus (HCV) protein translation is mediated by a cis-acting RNA, an internal ribosomal entry site (IRES), located in the 5' nontranslated region of the viral RNA. To examine proteins bound to the IRES, which could include proteins important for its function as well as potential drug targets, we used shotgun peptide sequencing to identify proteins in quadruplicate protein affinity extracts of lysed Huh7 cells, obtained using a biotinylated IRES. Twenty-six proteins bound the HCV IRES but not a reversed complementary sequence RNA or vector RNA controls. These included five ribosomal subunits, nine eukaryotic initiation factor 3 subunits, and novel interacting proteins such as the cytoskeletal-related proteins actin, FHOS (formin homologue overexpressed in spleen) and MIP-T3 (microtubule interacting protein that associates with TRAF3). Other novel HCV IRES-binding proteins included UNR (upstream of N-ras), UNR-interacting protein, and the RNA-binding proteins PAI-1 (plasminogen activator inhibitor-1) mRNA binding protein and Ewing sarcoma breakpoint 1 region protein EWS. A large set of additional proteins bound both the HCV IRES and a reversed complementary IRES sequence control, including the known HCV interactors PTB (polypyrimidine tract binding protein), the La autoantigen, and nucleolin. The discovery of these novel HCV IRES-binding proteins suggests links between IRES biology and the cytoskeleton, signal transduction, and other cellular functions.     

Changes in cytoskeletal organization in polyoma middle T antigen-transformed fibroblasts: involvement of protein phosphatase 2A and src tyrosine kinases

The major transforming activity of polyomavirus, middle T antigen, targets several cellular regulatory effectors including protein phosphatase 2A and src tyrosine kinases. Although transformed cells exhibit profound morphological changes, little is known about how middle T antigen-induced changes in the cellular regulatory environment specifically affect the cytoskeleton. We have investigated these changes in 10T(1/2) mouse fibroblasts transformed with polyoma middle T antigen. Immunofluorescence microscopy revealed that expression of middle T antigen (Pym T cells) depleted the stable (acetylated) microtubule array and increased the sensitivity of dynamic (tyrosinated) microtubules to nocodazole-induced disassembly. These effects were associated with a modest but statistically significant (P相似文献   

Mammalian homolog of the yeast cyclase associated protein, CAP/Srv2p, regulates actin filament assembly

Control of cell shape and motility requires rearrangements of the actin cytoskeleton. One cytoskeletal protein that may regulate actin dynamics is CAP (cyclase associated protein; CAP/Srv2p; ASP-56). CAP was first isolated from yeast as an adenylyl cyclase associated protein required for RAS regulation of cAMP signaling. In addition, CAP also regulates the actin cytoskeleton primarily through an actin monomer binding activity. CAP homologs are found in many eukaryotes, including mammals where they also bind actin, but little is known about their biological function. We, therefore, designed experiments to address CAP1 regulation of the actin cytoskeleton. CAP1 localized to membrane ruffles and actin stress fibers in fixed cells of various types. To address localization in living cells, we constructed GFP-CAP1 fusion proteins and found that fusion proteins lacking the actin-binding region localized like the wild type protein. We also performed microinjection studies with affinity-purified anti-CAP1 antibodies in Swiss 3T3 fibroblasts and found that the antibodies attenuated serum stimulation of stress fibers. Finally, CAP1 purified from platelets through a monoclonal antibody affinity purification step stimulated the formation of stress fiber-like filaments when it was microinjected into serum-starved Swiss 3T3 cells. Taken together, these data suggest that CAP1 promotes assembly of the actin cytoskeleton.     

Identification of Nd1, a novel murine kelch family protein,involved in stabilization of actin filaments

We isolated Nd1, a novel kelch family gene that encodes two forms of proteins, Nd1-L and Nd1-S. Nd1-L contains a BTB/POZ domain in its N terminus and six kelch repeats in the C terminus. Nd1-S has the BTB/POZ domain but lacks the six kelch repeats. Nd1-L but not Nd1-S mRNA is detected ubiquitously in normal mouse tissues. Nd1-L and Nd1-S proteins can form a dimer through the BTB/POZ domain. Nd1-L colocalizes with actin filaments detected using a confocal microscope, and its kelch repeats bind to them in vitro. Overexpression of Nd1-L in NIH3T3 cells delayed cell growth by affecting the transition of cytokinesis. Furthermore, the overexpression prevented NIH3T3 cells from cell death induced by actin destabilization but not by microtubule dysfunction. These data suggest that Nd1-L functions as a stabilizer of actin filaments as an actin-binding protein and may play a role in the dynamic organization of the actin cytoskeleton.     

Dynamics and regulation of plant interphase microtubules: a comparative view

Microtubule and actin cytoskeletons are fundamental to a variety of cellular activities within eukaryotic organisms. Extensive information on the dynamics and functions of microtubules, as well as on their regulatory proteins, have been revealed in fungi and animals, and corresponding pictures are now slowly emerging in plants. During interphase, plant cells contain highly dynamic cortical microtubules that organize into ordered arrays, which are apparently regulated by distinct groups of microtubule regulators. Comparison with fungal and animal microtubules highlights both conserved and unique mechanisms for the regulation of the microtubule cytoskeleton in plants.     

Rapid microtubule-dependent induction of neurite-like extensions in NIH 3T3 fibroblasts by inhibition of ROCK and Cbl

A number of key cellular functions, such as morphological differentiation and cell motility, are closely associated with changes in cytoskeletal dynamics. Many of the principal signaling components involved in actin cytoskeletal dynamics have been identified, and these have been shown to be critically involved in cell motility. In contrast, signaling to microtubules remains relatively uncharacterized, and the importance of signaling pathways in modulation of microtubule dynamics has so far not been established clearly. We report here that the Rho-effector ROCK and the multiadaptor proto-oncoprotein Cbl can profoundly affect the microtubule cytoskeleton. Simultaneous inhibition of these two signaling molecules induces a dramatic rearrangement of the microtubule cytoskeleton into microtubule bundles. The formation of these microtubule bundles, which does not involve signaling by Rac, Cdc42, Crk, phosphatidylinositol 3-kinase, and Abl, is sufficient to induce distinct neurite-like extensions in NIH 3T3 fibroblasts, even in the absence of microfilaments. This novel microtubule-dependent function that promotes neurite-like extensions is not dependent on net changes in microtubule polymerization or stabilization, but rather involves selective elongation and reorganization of microtubules into long bundles.     

The phosphorylation of p25/TPPP by LIM kinase 1 inhibits its ability to assemble microtubules

LIM kinase 1 (LIMK1) is a key regulator of actin dynamics as it phosphorylates and inactivates cofilin, an actin-depolymerizing factor. LIMK1 activity is also required for microtubule disassembly in endothelial cells. A search for LIMK1-interacting proteins identified p25alpha, a phosphoprotein that promotes tubulin polymerization. We found that p25 is phosphorylated by LIMK1 on serine residues in vitro and in cells. Immunoblotting analysis revealed that p25 is not a brain specific protein as previously reported, but is expressed in all mouse tissues. Immunofluorescence analysis demonstrated that endogenous p25 is co-localized with microtubules and is also found in the nucleus. Down-regulation of p25 by siRNA decreased microtubule levels while its overexpression in stable NIH-3T3 cell lines increased cell size and levels of stable tubulin. Bacterially expressed unphosphorylated p25 promotes microtubule assembly in vitro; however, when phosphorylated in cells, p25 lost its ability to assemble microtubule. Our results represent a surprising connection between the tubulin and the actin cytoskeleton mediated by LIMK1. We propose that the LIMK1 phosphorylation of p25 blocks p25 activity, thus promoting microtubule disassembly.     

Cytochalasin D enhances the accumulation of a protease‐resistant form of prion protein in ScN2a cells: involvement of PI3 kinase/Akt signalling pathway

The conversion of a host‐encoded PrPsen (protease‐sensitive cellular prion protein) into a PrPres (protease‐resistant pathogenic form) is a key process in the pathogenesis of prion diseases, but the intracellular mechanisms underlying PrPres amplification in prion‐infected cells remain elusive. To assess the role of cytoskeletal proteins in the regulation of PrPres amplification, the effects of cytoskeletal disruptors on PrPres accumulation in ScN2a cells that were persistently infected with the scrapie Chandler strain have been examined. Actin microfilament disruption with cytochalasin D enhanced PrPres accumulation in ScN2a cells. In contrast, the microtubule‐disrupting agents, colchicine, nocodazole and paclitaxel, had no effect on PrPres accumulation. In addition, a PI3K (phosphoinositide 3‐kinase) inhibitor, wortmannin and an Akt kinase inhibitor prevented the cytochalasin D‐induced enhancement of PrPres accumulation. Cytochalasin D‐induced extension of neurite‐like processes might correlate with enhanced accumulation of PrPres. The results suggest that the actin cytoskeleton and PI3K/Akt pathway are involved in the regulation of PrPres accumulation in prion‐infected cells.     

Mip1 associates with both the Mps1 kinase and actin,and is required for cell cortex stability and anaphase spindle positioning

The Mps1 family of protein kinases contributes to cell cycle control by regulating multiple microtubule cytoskeleton activities. We have uncovered a new Mps1 substrate that provides a novel link between Mps1 and the actin cytoskeleton. We have identified a conserved human Mps1 (hMps1) interacting protein we have termed Mps1 interacting protein-1 (Mip1). Mip1 defines an uncharacterized family of conserved proteins that contain coiled-coil and calponin homology domains. We demonstrate that Mip1 is a phosphoprotein that interacts with hMps1 in vitro and in vivo and is a hMps1 substrate. Mip1 exhibits dynamic localization during the cell cycle; Mip1 localizes to the actin cytoskeleton during interphase, the spindle in early mitosis, and the cleavage furrow during cytokinesis. Mip1 function is required to ensure proper spindle positioning at the onset of anaphase after cells begin furrow ingression. Cells depleted of Mip1 exhibit aberrant mitotic actin filament organization, excessive membrane blebbing, dramatic spindle rocking, and chromosome distribution errors during early cytokinesis producing high numbers of binucleate cells. Our data indicate that Mip1 is a newly recognized component of the actin cytoskeleton that interacts with hMps1 and that it is essential to ensure proper segregation of the genome during cell cleavage.     

Cytoskeleton reorganization in influenza hemagglutinin-initiated syncytium formation

Little is known about the mechanisms of cell-cell fusion in development and diseases and, especially, about fusion stages downstream of an opening of nascent fusion pore(s). Earlier works on different cell-cell fusion reactions have indicated that cytoskeleton plays important role in syncytium formation. However, due to complexity of these reactions and multifaceted contributions of cytoskeleton in cell physiology, it has remained unclear whether cytoskeleton directly drives fusion pore expansion or affects preceding fusion stages. Here we explore cellular reorganization associated with fusion pore expansion in syncytium formation using relatively simple experimental system. Fusion between murine embryonic fibroblasts NIH3T3-based cells is initiated on demand by well-characterized fusogen influenza virus hemagglutinin. We uncouple early fusion stages dependent on protein fusogens from subsequent fusion pore expansion stage and establish that the transition from local fusion to syncytium requires metabolic activity of living cells. Effective syncytium formation for cells with disorganized actin and microtubule cytoskeleton argues against hypothesis that cytoskeleton drives fusion expansion.     

Zebrafish lines expressing UAS‐driven red probes for monitoring cytoskeletal dynamics

Actin filaments and microtubules are principal components of the cytoskeleton that regulate the basic cellular phenomena underlying many fundamental cellular processes. Therefore, analyzing their dynamics in living cells is important for understanding cellular events more precisely. In this article, we report two novel transgenic zebrafish lines expressing red fluorescent proteins tagged with Lifeact or EB1 that interact with actin filaments and microtubule plus ends, respectively, under the control of the GAL4‐UAS system. Using these transgenic lines, we could detect F‐actin and microtubule plus end dynamics in specific tissues of living zebrafish embryos by crossing with GAL4 driver lines. In addition, we could achieve multi‐color imaging using these transgenic lines with GFP‐expressing transgenic lines. Therefore, our transgenic lines that carry UAS‐driven red fluorescent cytoskeletal probes are useful tools for analyzing spatiotemporal changes of the cytoskeletal elements using multicolor live imaging.     

Regulation of a formin complex by the microtubule plus end protein tea1p

The plus ends of microtubules have been speculated to regulate the actin cytoskeleton for the proper positioning of sites of cell polarization and cytokinesis. In the fission yeast Schizosaccharomyces pombe, interphase microtubules and the kelch repeat protein tea1p regulate polarized cell growth. Here, we show that tea1p is directly deposited at cell tips by microtubule plus ends. Tea1p associates in large "polarisome" complexes with bud6p and for3p, a formin that assembles actin cables. Tea1p also interacts in a separate complex with the CLIP-170 protein tip1p, a microtubule plus end-binding protein that anchors tea1p to the microtubule plus end. Localization experiments suggest that tea1p and bud6p regulate formin distribution and actin cable assembly. Although single mutants still polarize, for3Deltabud6Deltatea1Delta triple-mutant cells lack polarity, indicating that these proteins contribute overlapping functions in cell polarization. Thus, these experiments begin to elucidate how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth.     

Mip1 associates with both the Mps1 kinase and actin,and is required for cell cortex stability and anaphase spindle positioning

The Mps1 family of protein kinases contributes to cell cycle control by regulating multiple microtubule cytoskeleton activities. We have uncovered a new Mps1 substrate that provides a novel link between Mps1 and the actin cytoskeleton. We have identified a conserved human Mps1 (hMps1) interacting protein and have termed Mps1 interacting protein-1 (Mip1). Mip1 defines an uncharacterized family of conserved proteins that contain coiled-coil and calponin homology domains. We demonstrate that Mip1 is a phosphoprotein that interacts with hMps1 in vitro and in vivo and is a hMps1 substrate. Mip1 exhibits dynamic localization during the cell cycle; Mip1 localizes to the actin cytoskeleton during interphase, the spindle in early mitosis and the cleavage furrow during cytokinesis. Mip1 function is required to ensure proper spindle positioning at the onset of anaphase after cells begin furrow ingression. Cells depleted of Mip1 exhibit aberrant mitotic actin filament organization, excessive membrane blebbing, dramatic spindle rocking and chromosome distribution errors during early cytokinesis producing high numbers of binucleate cells. Our data indicate that Mip1 is a newly recognized component of the actin cytoskeleton that interacts with hMps1 and that it is essential to ensure proper segregation of the genome during cell cleavage.Key words: Mps1 kinase, actin, Mip1, cytokinesis     

TLRR (lrrc67) interacts with PP1 and is associated with a cytoskeletal complex in the testis

Background information. Spermatozoa are formed via a complex series of cellular transformations, including acrosome and flagellum formation, nuclear condensation and elongation and removal of residual cytoplasm. Nuclear elongation is accompanied by the formation of a unique cytoskeletal structure, the manchette. We have previously identified a leucine‐rich repeat protein that we have named TLRR (testis leucine‐rich repeat), associated with the manchette that contains a PP1 (protein phosphatase‐1)‐binding site. Leucine‐rich repeat proteins often mediate protein–protein interactions; therefore, we hypothesize that TLRR acts as a scaffold to link signalling molecules, including PP1, to the manchette near potential substrate proteins important for spermatogenesis. Results. TLRR and PP1 interact with one another as demonstrated by co‐immunoprecipitation and the yeast two‐hybrid assay. TLRR binds more strongly to PP1γ2 than it does to PP1α. Anti‐phosphoserine antibodies immunoprecipitate TLRR from testis lysate, indicating that TLRR is a phosphoprotein. TLRR is part of a complex in testis that includes cytoskeletal proteins and constituents of the ubiquitin–proteasome pathway. The TLRR complex purified from 3T3 cells contains similar proteins, co‐localizes with microtubules and is enriched at the microtubule‐organizing centre. TLRR is also detected near the centrosome of elongated, but not mid‐stage, spermatids. Conclusion. We demonstrate here that TLRR interacts with PP1, particularly the testis‐specific isoform, PP1γ2. Immunoaffinity purification confirms that TLRR is associated with the spermatid cytoskeleton. In addition, proteins involved in protein stability are part of the TLRR complex. These results support our hypothesis that TLRR links signalling molecules to the spermatid cytoskeleton in order to regulate important substrates involved in spermatid transformation. The translocation of TLRR from the manchette to the centrosome region suggests a possible role for this protein in tail formation. Our finding that TLRR is associated with microtubules in cultured cells suggests that TLRR may play a common role in modulating the cytoskeleton in other cell types besides male germ cells.     

Cytoskeleton/endoplasmic reticulum collapse induced by prostaglandin J2 parallels centrosomal deposition of ubiquitinated protein aggregates

Many neurodegenerative disorders, such as Parkinson disease, exhibit inclusion bodies containing ubiquitinated proteins. The mechanisms implicated in this aberrant protein deposition remain elusive. In these disorders signs of inflammation are also apparent in the affected central nervous system areas. We show that prostaglandin J2 (PGJ2), an endogenous product of inflammation, disrupts the cytoskeleton in neuronal cells. Furthermore, PGJ2 perturbed microtubule polymerization in vitro and decreased the number of free sulfhydryl groups on tubulin cysteines. A direct effect of PGJ2 on actin was not apparent, although actin filaments were altered in cells treated with PGJ2. This cyclopentenone prostaglandin triggered endoplasmic reticulum (ER) collapse and the redistribution of ER proteins, such as calnexin and catechol-O-methyltransferase, into a large centrosomal aggregate containing ubiquitinated proteins and alpha-synuclein. The PGJ2-dependent cytoskeletal rearrangement paralleled the development of the large centrosomal aggregate. Both of these events were replicated by treating cells with colchicine, which disrupts the microtubule/ER network, but not with brefeldin A, which impairs ER/Golgi transport. PGJ2 also perturbed 26 S proteasome assembly and activity, which preceded the accumulation of ubiquitinated proteins as detergent/salt-insoluble aggregates. Our data support a mechanism by which, upon PGJ2 treatment, cytoskeleton/ER collapse coincides with the relocation of ER proteins, other potentially neighboring proteins, and ubiquitinated proteins into centrosomal aggregates. Development of these large perinuclear aggregates is associated with disruption of the microtubule/ER network. This aberrant protein deposition, triggered by a product of inflammation, may be common to other compounds that disrupt microtubules and induce protein aggregation, such as MPP+ and rotenone, found to be associated with neurodegeneration.     

Nemitin, a novel Map8/Map1s interacting protein with Wd40 repeats

In neurons, a highly regulated microtubule cytoskeleton is essential for many cellular functions. These include axonal transport, regional specialization and synaptic function. Given the critical roles of microtubule-associated proteins (MAPs) in maintaining and regulating microtubule stability and dynamics, we sought to understand how this regulation is achieved. Here, we identify a novel LisH/WD40 repeat protein, tentatively named nemitin (neuronal enriched MAP interacting protein), as a potential regulator of MAP8-associated microtubule function. Based on expression at both the mRNA and protein levels, nemitin is enriched in the nervous system. Its protein expression is detected as early as embryonic day 11 and continues through adulthood. Interestingly, when expressed in non-neuronal cells, nemitin displays a diffuse pattern with puncta, although at the ultrastructural level it localizes along the microtubule network in vivo in sciatic nerves. These results suggest that the association of nemitin to microtubules may require an intermediary protein. Indeed, co-expression of nemitin with microtubule-associated protein 8 (MAP8) results in nemitin losing its diffuse pattern, instead decorating microtubules uniformly along with MAP8. Together, these results imply that nemitin may play an important role in regulating the neuronal cytoskeleton through an interaction with MAP8.