Microtubule-associated Protein 7 Research Articles

Resonance assignments of the microtubule-binding domain of the microtubule-associated protein 7 (MAP7)

The microtubule-associated protein 7 (MAP7) is a protein involved in cargo transport along microtubules (MTs) by interacting with kinesin-1 through the C-terminal kinesin-binding domain. Moreover, the protein is reported to stabilize MT, thereby playing a key role in axonal branch development. An important element for this latter function is the 112 amino-acid long N-terminal microtubule-binding domain (MTBD) of MAP7. Here we report NMR backbone and side-chain assignments that suggest a primarily alpha-helical secondary fold of this MTBD in solution. The MTBD contains a central long α-helical segment that includes a short four-residue ‘hinge’ sequence with decreased helicity and increased flexibility. Our data represent a first step towards analysing the complex interaction of MAP7 with MTs at an atomic level via NMR spectroscopy.

A Neurodevelopmental Disorder with Intellectual Disability and Autism due to MAP7D3 Haploinsufficiency

We report on a young boy with a neurodevelopmental disorder who is a carrier of a novel frameshift mutation in gene MAP7D3 (MAP7 domain containing 3) of Xq26.3. The protein encoded by this gene belongs to the MAP7 (microtubuleassociated protein 7) family that is proposed to regulate kinesin-1 (KIF5B)-dependent intracellular transport, by acting as Microtubule (MT)-tethered recruitment factors and activators of this kinesin [1-3]. And as other MT-associated proteins is conceivable the involvement of MAP7D3 in neuronal morphogenesis and therefore in neurodevelopmental disorders.

Synergistic autoinhibition and activation mechanisms control kinesin-1 motor activity.

SUMMARYKinesin-1 activity is regulated by autoinhibition. Intramolecular interactions within the kinesin heavy chain (KHC) are proposed to be one facet of motor regulation. The KHC also binds to the kinesin light chain (KLC), which has been implicated in both autoinhibition and activation of the motor. We show that the KLC inhibits the kinesin-microtubule interaction independently from the proposed intramolecular interaction within KHC. Cargo-adaptor proteins that bind the KLC stimulated processive movement, but the landing rate of activated kinesin complexes remained low. Microtubule-associated protein 7 (MAP7) enhanced motility by increasing the landing rate and run length of the activated kinesin motors. Our results support a model whereby the motor activity of the kinesin is regulated by synergistic inhibition mechanisms and that cargo-adaptor binding to the KLC releases both mechanisms. However, a non-motor MAP is required for robust microtubule association of the activated motor. Thus, human kinesin is regulated by synergistic autoinhibition and activation mechanisms.

The kinesin-1 regulation mechanism of MAP7

Kinesin-1 is an essential molecular motor that performs microtubule (MT) based transport of cargos in the cell. MT associated protein 7 (MAP7) is a required cofactor to kinesin-1 shown to promote motor processivity and is responsible for cargo transport and organelle positioning. Using in vitro reconstitution methods, protein engineering, and single molecule imaging, we dissected the mechanism by which MAP7 influences kinesin activity. MAP7 recruits kinesin-1 to the MT through a kinesin-binding domain in its intrinsically disordered projection region.

Insulin-like growth factor-1 regulates the mechanosensitivity of chondrocytes by modulating TRPV4

Both mechanical and biochemical stimulation are required for maintaining the integrity of articular cartilage. However, chondrocytes respond differently to mechanical stimuli in osteoarthritic cartilage when biochemical signaling pathways, such as Insulin-like Growth Factor-1 (IGF-1), are altered. The Transient Receptor Potential Vanilloid 4 (TRPV4) channel is central to chondrocyte mechanotransduction and regulation of cartilage homeostasis. Here, we propose that changes in IGF-1 can modulate TRPV4 channel activity. We demonstrate that physiologic levels of IGF-1 suppress hypotonic-induced TRPV4 currents and intracellular calcium flux by increasing apparent cell stiffness that correlates with actin stress fiber formation. Disruption of F-actin following IGF-1 treatment results in the return of the intracellular calcium response to hypotonic swelling. Using point mutations of the TRPV4 channel at the microtubule-associated protein 7 (MAP-7) site shows that regulation of TRPV4 by actin is mediated via the interaction of actin with the MAP-7 domain of TRPV4. We further highlight that ATP release, a down-stream response to mechanical stimulation in chondrocytes, is mediated by TRPV4 during hypotonic challenge. This response is significantly abrogated with IGF-1 treatment. As chondrocyte mechanosensitivity is greatly altered during osteoarthritis progression, IGF-1 presents as a promising candidate for prevention and treatment of articular cartilage damage.

Plasma Exosomal CircNEK9 Accelerates the Progression of Gastric Cancer via miR-409-3p/MAP7 Axis.

Exosome-mediated transfer of circular RNAs (circRNAs) is related to gastric cancer (GC) development. CircRNA NIMA-related kinase 9 (circNEK9; hsa_circ_0032683) was reported to be up-regulated in GC. The biological role of circNEK9 and its underlying mechanisms in GC progression were explored. The levels of RNAs and proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assay. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and flow cytometry. Wound healing assay and transwell assays were conducted to analyze cell motility. Intermolecular interaction was verified by dual-luciferase reporter assay and RNA pull-down assay. Animal experiments were used to evaluate the role of circNEK9 in the growth of xenograft tumors in vivo. CircNEK9 was up-regulated in GC tissues and cell lines. CircNEK9 interference suppressed the proliferation and motility of GC cells. CircNEK9 silencing enhanced microRNA-409-3p (miR-409-3p) level through direct interaction. CircNEK9 silencing-mediated influences on the proliferation and metastasis of GC cells were partly overturned by the interference of miR-409-3p. MiR-409-3p directly interacted with microtubule-associated protein 7 (MAP7) messenger RNA (mRNA). MiR-409-3p-induced effects in GC cells were largely counteracted by the overexpression of MAP7. CircNEK9 silencing blocked GC tumor growth in vivo. Exosome-mediated transfer of circNEK9 promoted the motility of recipient GC cells. CircNEK9 accelerated the proliferation, migration, and invasion of GC cells through targeting miR-409-3p/MAP7 axis. Plasma exosomal circNEK9 promoted the migration and invasion of recipient GC cells.

Enhanced expression of microtubule-associated protein 7 functioned as a contributor to cervical cancer cell migration and is predictive of adverse prognosis

BackgroundCervical cancer (CC) is one of the most common female malignancies over the world. Microtubule-associated protein 7 (MAP7) belongs to the family of microtubule-associated proteins (MAPs) which involve in microtubule dynamics and are critical in several important cellular and intracellular activities. This study aimed to investigate the expression and potential role of MAP7 in CC.MethodsThe expression level of MAP7 in CC tissues and normal tissues were analyzed using the data obtained from The cancer genomes atlas (TCGA) and genotype-tissue expression (GTEx) databases. The prognostic value of MAP7 in patients with CC was analyzed by Kaplan–Meier analysis, Univariate and Multivariate analyses. Moreover, the influences of MAP7 expression alteration on the viability and motility of Caski, HeLa and C-33A cells was measured by CCK8 assay, colony formation assay, scratch assay, and transwell migration and invasion assays. Flow cytometry was conducted to determine cell apoptosis. Western blot was performed to evaluate the impact of MAP7 on the expression of apoptotic-related proteins as well as mitogen-activated protein kinase (MAPK) signaling pathway-related proteins. In vivo tumorigenicity assay was performed to explore the influence of MAP7 on tumor growth.ResultsUp-regulation of MAP7 was observed in CC tissues and high MAP7 expression was positively correlated with worse prognosis. Multivariate analyses suggested that MAP7 expression can be served as an independent predictor for overall survival of patients with CC. Knockdown of MAP7 markedly suppressed Caski and HeLa cell viability, migration and invasion while notably induced cell apoptosis. Furthermore, depletion of MAP7 in Caski and HeLa cells elevated the expression levels of Active-caspase 3 and Bax, but declined the level of Bcl-2. Whilst, overexpression of MAP7 in C-33A cells presented the opposite outcomes. Additionally, knockdown of MAP7 significantly decreased the phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) in Caski and HeLa cells, and overexpression of MAP7 increased their phosphorylation in C-33A cells, indicating that MAP7 may regulate the MAPK signaling pathway in CC cells. In vivo assays revealed that knockdown of MAP7 remarkably repressed the growth of CC tumors.ConclusionThe results of the present study suggest that MAP7 functions as a promoter during the occurrence and progression of CC, and that MAP7 may serve as a promising therapeutic target in CC.

Characterization and Evaluation of Sunitinib and Janus Kinase Inhibitors in a Renal Cell Carcinoma Cell Line, Caki‐2

Kidney cancer is one of the leading cancers detected in middle‐aged men and women. The clear‐cell form of renal cell carcinoma (RCC) is the deadliest form of urogenital cancer; 5‐year prognosis once metastasis has occurred is only 5%. The main challenge of RCC diagnosis is that most symptoms of kidney cancer only appear after it has advanced to metastasis, the most common subtype being clear cell renal carcinoma (ccRCC). At this point, the cancer may have already gained resistance to current treatments. One intracellular signaling pathway, the Janus kinase 2 and signal transducers and activator of transcription 1 and 3 (JAK2/STAT1&3) pathway, has already been implicated in multiple other cancers and is involved in perpetuating many hallmarks of cancer including evasion of apoptosis, angiogenesis, tissue invasion, and metastasis. Microtubule associated protein 7 (MAP7) is a protein involved in tubulin and microtubule stability. High MAP7 expression has been correlated with poor prognosis in colon cancer. Additionally, MAP7 has been shown to be involved in chemo resistance via a STAT1 mechanism. Therefore, we hypothesized that increased MAP7 expression via increased JAK/STAT signaling is a cause of increased chemo resistance and cell proliferation in RCC. Therefore, inhibition of this pathway should increase efficacy of current chemotherapies. Available drugs demonstrate different molecular and physical reactions in RCC cells such as those that effect the cancer promoting unregulated JAK/STAT pathway. MTT analysis of the commercial ccRCC cell line, Caki‐2, with a current chemotherapy drug, sunitinib, shows increased cell viability prior to reaching its EC50 value at 24, 48, and 72 hours of 30 uM, 16.5 uM, and 12 uM, respectively, suggesting therapy resistant mechanisms are taking place. When the known JAK inhibitor Zerumbone was evaluated in Caki‐2 via MTT analysis, EC50 values were 175 uM, 32 uM, and 15 uM at 24, 48, and 72 hours, respectively, with increased cell viability observed only in the 24 hour assay. When these two drugs are used in combination with Caki‐2, MTT data suggests resistance to sunitinib and zerumbone exists in Caki‐2 and co‐treatment aided the ability to decrease cell viability by sunitinib, but not Zerumbone. Based on these results, more studies must be completed to determine whether the inhibition of JAK would be a viable and effective option in treatment of RCC. However, preliminary evaluation of Zerumbone in combination with sunitinib has not proven to be effective.Support or Funding InformationOakland University Center For Biomedical Research, Zafarna Grant, Oakland University Honors CollegeThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P5-05-01: Physico-biochemical regulation of EMT by microtubule associated protein 7 (MAP7)

Abstract Background: We previously reported about 500 cancer stem cell (CSC) specific gene signatures from patient tumor samples. After screening with shRNAs for the 500 genes affecting mammosphere forming ability, we identified microtubule-assoicated protein 7 (MAP7) as one of the top candidate genes, which may serve as a target for breast CSCs. Although MAP7 is a predominant epithelial microtubule binding protein, only limited number of reports suggests that MAP7 may be a regulator of microtubule dynamics during cell division and a cofactor of Kinesin-1 in compartment transport in cells. However, little is known about how MAP7 supports epithelial cancers, especially breast cancer. Previously, we have reported that the mammosphere forming cells exhibit treatment resistance and high metastatic potential, which are intrinsic characters for CSCs displaying epithelial mesenchymal transition (EMT). We hypothesize that MAP7 supports breast cancer progression by promoting CSC self-renewal and survival through regulation of EMT. Objectives: Here, we aim to show that MAP7 is an essential regulator of breast CSCs and to elucidate mechanism behind EMT regulation by MAP7 in breast CSCs. Methods and Results: On Oncomine database analysis, MAP7 was up-regulated in most epithelial cancers, when compared to the corresponding normal tissues. Similarly, its expression in breast cancer was 2-fold higher than in the normal breast tissue (p<0.05), but without significant variances in the expression across the breast cancer subtypes. Gene silencing of MAP7 significantly reduced CD44+/CD24- breast CSC populations and mammosphere forming efficiencies of MDA-MB-231, HCC1937, and MDA-MB-468 breast cancer cells. Furthermore, the silencing of MAP7 expression compromised invasive potential of MDA-MB-231 cells by 50% and significantly altered the cell membrane mechanics of MDA-MB-468 cells, as indicated by a high-content image analysis for cell shapes and cell adhesion efficiency. More importantly, delivery of siRNA in vivo inhibited the growth of BCM2147 patient-derived tumor, and limiting dilution assay demonstrated that the tumor initiation potential of BCM2147 can be eliminated by MAP7 silencing. Through confocal microscope analysis of images of fluorescent immunostaining and co-immunoprecipatation assays, MAP7 showed polarized-expressions in spindle-shaped cancer cells and was co-localized with Focal Adhesion Kinase (FAK). Moreover, MAP7 silencing inhibited the phosphorylation of FAK by inactivating p130CAS and JSAP1, the upper stream and the down-stream regulators of FAK. Conclusion: We have showed the ectopic expression of MAP7 in breast tumors and other epithelial tumors, suggesting MAP7 may be involved in tumorigenesis and critical for the survival of tumor cells. Moreover, our results suggest that MAP7 is a key element for survival and self-renewal of breast CSCs through polarization of cells and activation of FAK, required for the initiation of EMT. To that end, here we report that MAP7 is essential for breast cancer growth by supporting CSC survival and self-renewal. Citation Format: Choi DS, Dave B, Rosato RR, Chang JC. Physico-biochemical regulation of EMT by microtubule associated protein 7 (MAP7) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-05-01.

MAP7 Regulates Axon Collateral Branch Development in Dorsal Root Ganglion Neurons.

Collateral branches from axons are key components of functional neural circuits that allow neurons to connect with multiple synaptic targets. Like axon growth and guidance, formation of collateral branches depends on the regulation of microtubules, but how such regulation is coordinated to ensure proper circuit development is not known. Based on microarray analysis, we have identified a role for microtubule-associated protein 7 (MAP7) during collateral branch development of dorsal root ganglion (DRG) sensory neurons. We show that MAP7 is expressed at the onset of collateral branch formation. Perturbation of its expression by overexpression or shRNA knockdown alters axon branching in cultured DRG neurons. Localization and time-lapse imaging analysis reveals that MAP7 is enriched at branch points and colocalizes with stable microtubules, but enters the new branch with a delay, suggesting a role in branch maturation. We have also investigated a spontaneous mutant mouse that expresses a truncated MAP7 and found a gain-of-function phenotype both in vitro and in vivo Further domain analysis suggests that the amino half of MAP7 is responsible for branch formation, suggesting a mechanism that is independent of its known interaction with kinesin. Moreover, this mouse exhibits increased pain sensitivity, a phenotype that is consistent with increased collateral branch formation. Therefore, our study not only uncovers the first neuronal function of MAP7, but also demonstrates the importance of proper microtubule regulation in neural circuit development. Furthermore, our data provide new insights into microtubule regulation during axonal morphogenesis and may shed light on MAP7 function in neurological disorders.SIGNIFICANCE STATEMENT Neurons communicate with multiple targets by forming axonal branches. In search of intrinsic factors that control collateral branch development, we identified a role for microtubule-associated protein 7 (MAP7) in dorsal root ganglion sensory neurons. We show that MAP7 expression is developmentally regulated and perturbation of this expression alters branch formation. Cell biological analysis indicates that MAP7 promotes branch maturation. Analysis of a spontaneous mouse mutant suggests a molecular mechanism for branch regulation and the potential influence of collateral branches on pain sensitivity. Our studies thus establish the first neuronal function of MAP7 and demonstrate its role in branch morphogenesis and neural circuit function. These findings may help in our understanding of the contribution of MAP7 to neurological disorders and nerve regeneration.

High expression of MAP7 predicts adverse prognosis in young patients with cytogenetically normal acute myeloid leukemia.

Microtubule-associated protein 7 (MAP7) plays an important role in cancer cells. In this study, we identified the prognostic significance of MAP7 expression in cytogenetically normal acute myeloid leukemia (CN-AML) patients (aged <60 years) based on several microarray datasets. In the first group (n = 129), high MAP7 expression (MAP7high) was associated with adverse overall survival (OS; P = 0.0441) and event-free survival (EFS; P = 0.0114) compared with low MAP7 expression (MAP7low). In addition, the prognostic significance of MAP7 was confirmed by European Leukemia Net (ELN) intermediate-I genetic categories and multivariable analysis. In the second independent group of CN-AML patients (aged <60 years), MAP7high was also associated with adverse OS (n = 88, OS; P = 0.00811). To understand the inherent mechanisms of MAP7’s prognosis, we investigated genome-wide gene/microRNA expression signatures associated with MAP7 expression. Several known oncogenic genes/microRNAs and anti-oncogenic genes/microRNAs were disordered in MAP7high CN-AML patients. In conclusion, MAP7high is an adverse prognostic biomarker for CN-AML, which may be attributed to the distinctive genome-wide gene/microRNA expression and related cell signaling pathways.

C-terminal region of MAP7 domain containing protein 3 (MAP7D3) promotes microtubule polymerization by binding at the C-terminal tail of tubulin.

MAP7 domain containing protein 3 (MAP7D3), a newly identified microtubule associated protein, has been shown to promote microtubule assembly and stability. Its microtubule binding region has been reported to consist of two coiled coil motifs located at the N-terminus. It possesses a MAP7 domain near the C-terminus and belongs to the microtubule associated protein 7 (MAP7) family. The MAP7 domain of MAP7 protein has been shown to bind to kinesin-1; however, the role of MAP7 domain in MAP7D3 remains unknown. Based on the bioinformatics analysis of MAP7D3, we hypothesized that the MAP7 domain of MAP7D3 may have microtubule binding activity. Indeed, we found that MAP7 domain of MAP7D3 bound to microtubules as well as enhanced the assembly of microtubules in vitro. Interestingly, a longer fragment MDCT that contained the MAP7 domain (MD) with the C-terminal tail (CT) of the protein promoted microtubule polymerization to a greater extent than MD and CT individually. MDCT stabilized microtubules against dilution induced disassembly. MDCT bound to reconstituted microtubules with an apparent dissociation constant of 3.0±0.5 µM. An immunostaining experiment showed that MDCT localized along the length of the preassembled microtubules. Competition experiments with tau indicated that MDCT shares its binding site on microtubules with tau. Further, we present evidence indicating that MDCT binds to the C-terminal tail of tubulin. In addition, MDCT could bind to tubulin in HeLa cell extract. Here, we report a microtubule binding region in the C-terminal region of MAP7D3 that may have a role in regulating microtubule assembly dynamics.

The identification of novel targets of miR-16 and characterization of their biological functions in cancer cells

BackgroundIn eukaryotes, miR-16 is an important microRNA (miRNA) that is involved in numerous biological processes. However, it is not fully understood how miR-16 executes its physiological functions. In the present study, we aimed to identify novel miR-16 targets and study their biological functions.MethodsCandidate target genes of miR-16 were screened by microarray analysis of mRNA levels in several cancer cell lines with enhanced miR-16. Three bioinformatics algorithms, including TargetScan, PicTar, and miRanda, were used in combination to calculate the miR-16 targets. The expression levels of miR-16 and target mRNA were examined by relative quantification RT-PCR, and the expression levels of target protein were detected by Western blot. Luciferase reporter plasmids were constructed to confirm direct targeting. The effect of miR-16 and target gene on cell viability was evaluated using MTT assays. The effects of miR-16 and target gene on apoptosis and cell cycle distribution were evaluated by flow cytometry analysis.ResultsBy overexpressing miR-16 in several cancer cell lines and measuring global mRNA levels using microarray analysis, we identified 27 genes that may be regulated by miR-16. After the bioinformatics filtering process, 18 genes were selected as candidate miR-16 targets. Furthermore, we experimentally validated three of these candidates, MAP7 (microtubule-associated protein 7), PRDM4 (PR domain containing 4) and CDS2 (CDP-diacylglycerol synthase 2), as direct targets of miR-16. Finally, we demonstrated that miR-16 targeting MAP7 played a critical role in regulating proliferation but not apoptosis and cell cycle progression in cancer cells.ConclusionIn summary, the present study identifies several novel miR-16 targets and illustrates a novel function of miR-16 targeting MAP7 in modulating proliferation in cancer cells.

MAP and kinesin-dependent nuclear positioning is required for skeletal muscle function

The basic unit of skeletal muscle in all metazoans is the multinucleate myofiber, within which individual nuclei are regularly positioned1. The molecular machinery responsible for myonuclear positioning is not known. Improperly positioned nuclei are a hallmark of numerous muscles diseases2, including centronuclear myopathies3, but it is unclear whether correct nuclear positioning is necessary for muscle function. Here we identify the microtubule-associated protein Ensconsin(Ens)/MAP7 and Kinesin Heavy Chain (Khc)/Kif5b as essential, evolutionary conserved regulators of myonuclear positioning in Drosophila and cultured mammalian myotubes. We find that these proteins physically interact and that expression of the Kif5b motor domain fused to the MAP7 microtubule binding domain rescues nuclear positioning defects in MAP7 depleted cells. This suggests that MAP7 links Kif5b to the microtubule cytoskeleton to promote nuclear positioning. Finally we demonstrate that myonuclear positioning is physiologically important. Drosophila ens mutant larvae display decreased locomotion and incorrect myonuclear positioning, and these phenotypes are rescued by muscle specific expression of Ens. We conclude that improper nuclear positioning contributes to muscle dysfunction in a cell autonomous fashion.

Mdp3 is a novel microtubule-binding protein that regulates microtubule assembly and stability

Microtubule-binding proteins are a group of molecules that associate with microtubules, regulate the structural properties of microtubules, and thereby participate in diverse microtubule-mediated cellular activities. A recent mass spectrometry-based proteomic study has identified microtubule-associated protein 7 (MAP7) domain-containing 3 (Mdp3) as a potential microtubule-binding protein. However, its subcellular localization and functional importance are not characterized. In this study, by GST-pulldown assays, we found that Mdp3 interacted with tubulin both in cells and in vitro. Immunofluorescence microscopy and microtubule cosedimentation assays revealed that Mdp3 also associated with microtubules. Serial deletion experiments showed that the two coiled coil motifs of Mdp3 were critical for its interaction with tubulin and microtubules. Cold recovery and nocodazole washout assays further demonstrated an important role for Mdp3 in regulating cellular microtubule assembly. Our data also showed that Mdp3 significantly enhanced the stability of cellular microtubules. By tubulin turbidity assay, we found that Mdp3 could promote microtubule assembly and stability in the purified system. In addition, we found that Mdp3 expression varied during the cell cycle and in primary tissues. These findings thus establish Mdp3 as a novel microtubule-binding protein that regulates microtubule assembly and stability.

Microfilament-associated Protein 7 Increases the Membrane Expression of Transient Receptor Potential Vanilloid 4 (TRPV4)

The molecular mechanism of the transmission of changes in the shape of the cell surface to ion channels remains obscure. Ca2+ influx induced by cell deformity is inhibited by actin-freezing reagents, suggesting that the actin microfilament couples with an ion channel. Transient receptor potential vanilloid 4 (TRPV4) is a candidate in the calcium-permeable, swelling-activated mechanosensitive channel in heterogeneously expressed cells. To investigate the mechanosensitive molecular complex, we found that microtubule-associated protein 7 (MAP7) is the mouse TRPV4 C-terminal binding protein. MAP7 was coimmunoprecipitated with TRPV4. The results of a pull-down assay demonstrated that the alignment of amino acids 798-809 of TRPV4 was important in this interaction. TRPV4 and MAP7 colocalized in the lung and kidney. The coexpression of these two molecules resulted in the redistribution of TRPV4 toward the membrane and increased its functional expression. The alignment of amino acids 798-809 of TRPV4 was also important in the functional expression. The activated current was abolished by actin-freezing but not by microtubule-freezing reagents. We therefore believe that MAP7 may enhance the membrane expression of TRPV4 and possibly link cytoskeletal microfilaments.