Identification of the Isoform-specific Interactions between the Tail and the Head of Class V Myosin

Vertebrates have three isoforms of class V myosin (Myo5), Myo5a, Myo5b, and Myo5c, which are involved in transport of multiple cargoes. It is well established that the motor functions of Myo5a and Myo5b are regulated by a tail inhibition mechanism. Here we found that the motor function of Myo5c was also inhibited by its globular tail domain (GTD), and this inhibition was abolished by high Ca²⁺, indicating that the tail inhibition mechanism is conserved in vertebrate Myo5. Interestingly, we found that Myo5a-GTD and Myo5c-GTD were not interchangeable in terms of inhibition of motor function, indicating isoform-specific interactions between the GTD and the head of Myo5. To identify the isoform-specific interactions, we produced a number of Myo5 chimeras by swapping the corresponding regions of Myo5a and Myo5c. We found that Myo5a-GTD, with its H11-H12 loop being substituted with that of Myo5c, was able to inhibit the ATPase activity of Myo5c and that Myo5a-GTD was able to inhibit the ATPase activity of Myo5c-S1 and Myo5c-HMM only when their IQ1 motif was substituted with that of Myo5a. Those results indicate that the H11-H12 loop in the GTD and the IQ1 motif in the head dictate the isoform-specific interactions between the GTD and head of Myo5. Because the IQ1 motif is wrapped by calmodulin, whose conformation is influenced by the sequence of the IQ1 motif, we proposed that the calmodulin bound to the IQ1 motif interacts with the H11-H12 loop of the GTD in the inhibited state of Myo5.     

Motor protein Myo1c is a podocyte protein that facilitates the transport of slit diaphragm protein Neph1 to the podocyte membrane

The podocyte proteins Neph1 and nephrin organize a signaling complex at the podocyte cell membrane that forms the structural framework for a functional glomerular filtration barrier. Mechanisms regulating the movement of these proteins to and from the membrane are currently unknown. This study identifies a novel interaction between Neph1 and the motor protein Myo1c, where Myo1c plays an active role in targeting Neph1 to the podocyte cell membrane. Using in vivo and in vitro experiments, we provide data supporting a direct interaction between Neph1 and Myo1c which is dynamic and actin dependent. Unlike wild-type Myo1c, the membrane localization of Neph1 was significantly reduced in podocytes expressing dominant negative Myo1c. In addition, Neph1 failed to localize at the podocyte cell membrane and cell junctions in Myo1c-depleted podocytes. We further demonstrate that similarly to Neph1, Myo1c also binds nephrin and reduces its localization at the podocyte cell membrane. A functional analysis of Myo1c knockdown cells showed defects in cell migration, as determined by a wound assay. In addition, the ability to form tight junctions was impaired in Myo1c knockdown cells, as determined by transepithelial electric resistance (TER) and bovine serum albumin (BSA) permeability assays. These results identify a novel Myo1c-dependent molecular mechanism that mediates the dynamic organization of Neph1 and nephrin at the slit diaphragm and is critical for podocyte function.     

Activation of RalA is required for insulin-stimulated Glut4 trafficking to the plasma membrane via the exocyst and the motor protein Myo1c

Insulin stimulates glucose transport in muscle and adipose tissue by producing translocation of the glucose transporter Glut4. The exocyst, an evolutionarily conserved vesicle tethering complex, is crucial for targeting Glut4 to the plasma membrane. Here we report that insulin regulates this process via the G protein RalA, which is present in Glut4 vesicles and interacts with the exocyst in adipocytes. Insulin stimulates the activity of RalA in a PI 3-kinase-dependent manner. Disruption of RalA function by dominant-negative mutants or siRNA-mediated knockdown attenuates insulin-stimulated glucose transport. RalA also interacts with Myo1c, a molecular motor implicated in Glut4 trafficking. This interaction is modulated by Calmodulin, which functions as the light chain for Myo1c during insulin-stimulated glucose uptake. Thus, RalA serves two functions in insulin action: as a cargo receptor for the Myo1c motor, and as a signal for the unification of the exocyst to target Glut4 vesicles to the plasma membrane.     

Calcium, nucleotide, and actin affect the interaction of mammalian Myo1c with its light chain calmodulin

To investigate the interaction of mammalian class I myosin, Myo1c, with its light chain calmodulin, we expressed (with calmodulin) truncation mutants consisting of the Myo1c motor domain followed by 0-4 presumed calmodulin-binding (IQ) domains (Myo1c (0IQ)-Myo1c (4IQ)). The amount of calmodulin associating with the Myo1c heavy chain increased with increasing number of IQ domains from Myo1c (0IQ) to Myo1c (3IQ). No calmodulin beyond that associated with Myo1c (3IQ) was found with Myo1c (4IQ) despite its availability, showing that Myo1c binds three molecules of calmodulin with no evidence of a fourth IQ domain. Unlike Myo1c (0IQ), the basal ATPase activity of Myo1c (1IQ) was >10-fold higher in Ca (2+) vs EGTA +/- exogenous calmodulin, showing that regulation is by Ca (2+) binding to calmodulin on the first IQ domain. The K m and V max of the actin-activated Mg (2+)-ATPase activity were largely independent of the number of IQ domains present and moderately affected by Ca (2+). In binding assays, some calmodulin pelleted with Myo1c heavy chain when actin was present, but a considerable fraction remained in the supernatant, suggesting that calmodulin is displaced most likely from the second IQ domain. The Myo1c heavy chain associated with actin in a nucleotide-dependent fashion. In ATP a smaller proportion of calmodulin pelleted with the heavy chain, suggesting that Myo1c undergoes nucleotide-dependent conformational changes that affect the affinity of calmodulin for the heavy chain. The studies support a model in which Myo1c in the inner ear is regulated by both Ca (2+) and nucleotide, which exert their effects on motor activity through the light-chain-binding region.     

Myosin motor Myo1c and its receptor NEMO/IKK-gamma promote TNF-alpha-induced serine307 phosphorylation of IRS-1

Tumor necrosis factor-alpha (TNF-alpha) signaling through the IkappaB kinase (IKK) complex attenuates insulin action via the phosphorylation of insulin receptor substrate 1 (IRS-1) at Ser307. However, the precise molecular mechanism by which the IKK complex phosphorylates IRS-1 is unknown. In this study, we report nuclear factor kappaB essential modulator (NEMO)/IKK-gamma subunit accumulation in membrane ruffles followed by an interaction with IRS-1. This intracellular trafficking of NEMO requires insulin, an intact actin cytoskeletal network, and the motor protein Myo1c. Increased Myo1c expression enhanced the NEMO-IRS-1 interaction, which is essential for TNF-alpha- induced phosphorylation of Ser307-IRS-1. In contrast, dominant inhibitory Myo1c cargo domain expression diminished this interaction and inhibited IRS-1 phosphorylation. NEMO expression also enhanced TNF-alpha-induced Ser307-IRS-1 phosphorylation and inhibited glucose uptake. In contrast, a deletion mutant of NEMO lacking the IKK-beta-binding domain or silencing NEMO blocked the TNF-alpha signal. Thus, motor protein Myo1c and its receptor protein NEMO act cooperatively to form the IKK-IRS-1 complex and function in TNF-alpha-induced insulin resistance.     

Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles

GLUT4-containing vesicles cycle between the plasma membrane and intracellular compartments. Insulin promotes GLUT4 exocytosis by regulating GLUT4 vesicle arrival at the cell periphery and its subsequent tethering, docking, and fusion with the plasma membrane. The molecular machinery involved in GLUT4 vesicle tethering is unknown. We show here that Myo1c, an actin-based motor protein that associates with membranes and actin filaments, is required for insulin-induced vesicle tethering in muscle cells. Myo1c was found to associate with both mobile and tethered GLUT4 vesicles and to be required for vesicle capture in the total internal reflection fluorescence (TIRF) zone beneath the plasma membrane. Myo1c knockdown or overexpression of an actin binding–deficient Myo1c mutant abolished insulin-induced vesicle immobilization, increased GLUT4 vesicle velocity in the TIRF zone, and prevented their externalization. Conversely, Myo1c overexpression immobilized GLUT4 vesicles in the TIRF zone and promoted insulin-induced GLUT4 exposure to the extracellular milieu. Myo1c also contributed to insulin-dependent actin filament remodeling. Thus we propose that interaction of vesicular Myo1c with cortical actin filaments is required for insulin-mediated tethering of GLUT4 vesicles and for efficient GLUT4 surface delivery in muscle cells.     

Structural Insights into the Globular Tails of the Human Type V Myosins Myo5a,Myo5b,and Myo5c

Vertebrate type V myosins (MyoV) Myo5a, Myo5b, and Myo5c mediate transport of several different cargoes. All MyoV paralogs bind to cargo complexes mainly by their C-terminal globular domains. In absence of cargo, the globular domain of Myo5a inhibits its motor domain. Here, we report low-resolution SAXS models for the globular domains from human Myo5a, Myo5b, and Myo5c, which suggest very similar overall shapes of all three paralogs. We determined the crystal structures of globular domains from Myo5a and Myo5b, and provide a homology model for human Myo5c. When we docked the Myo5a crystal structure into a previously published electron microscopy density of the autoinhibited full-length Myo5a, only one domain orientation resulted in a good fit. This structural arrangement suggests the participation of additional region of the globular domain in autoinhibition. Quantification of the interaction of the Myo5a globular domain with its motor complex revealed a tight binding with dissociation half-life in the order of minutes, suggesting a rather slow transition between the active and inactive states.     

Human deafness mutation E385D disrupts the mechanochemical coupling and subcellular targeting of myosin-1a

Missense mutations in the membrane-binding actin-based motor protein, myosin-1a (Myo1a), have recently been linked to sensorineural deafness in humans. One of these mutations, E385D, impacts a residue in the switch II region of the motor domain that is present in virtually all members of the myosin superfamily. We sought to examine the impact of E385D on the function of Myo1a, both in terms of mechanochemical activity and ability to target to actin-rich microvilli in polarized epithelial cells. While E385D-Myo1a demonstrated actin-activated ATPase activity, the V_MAX was reduced threefold relative to wild-type. Despite maintaining an active mechanochemical cycle, E385D-Myo1a was unable to move actin in the sliding filament assay. Intriguingly, when an enhanced-green-fluorescent-protein-tagged form of E385D-Myo1a was stably expressed in polarized epithelial cells, this mutation abolished the microvillar targeting normally demonstrated by wild-type Myo1a. Notably, these data are the first to suggest that mechanical activity is essential for proper localization of Myo1a in microvilli. These studies also provide a unique example of how even the most mild substitution of invariant switch II residues can effectively uncouple enzymatic and mechanical activity of the myosin motor domain.     

Myosin 1G Is an Abundant Class I Myosin in Lymphocytes Whose Localization at the Plasma Membrane Depends on Its Ancient Divergent Pleckstrin Homology (PH) Domain (Myo1PH)

Class I myosins, which link F-actin to membrane, are largely undefined in lymphocytes. Mass spectrometric analysis of lymphocytes identified two short tail forms: (Myo1G and Myo1C) and one long tail (Myo1F). We investigated Myo1G, the most abundant in T-lymphocytes, and compared key findings with Myo1C and Myo1F. Myo1G localizes to the plasma membrane and associates in an ATP-releasable manner to the actin-containing insoluble pellet. The IQ+tail region of Myo1G (Myo1C and Myo1F) is sufficient for membrane localization, but membrane localization is augmented by the motor domain. The minimal region lacks IQ motifs but includes: 1) a PH-like domain; 2) a “Pre-PH” region; and 3) a “Post-PH” region. The Pre-PH predicted α helices may contribute electrostatically, because two conserved basic residues on one face are required for optimal membrane localization. Our sequence analysis characterizes the divergent PH domain family, Myo1PH, present also in long tail myosins, in eukaryotic proteins unrelated to myosins, and in a probable ancestral protein in prokaryotes. The Myo1G Myo1PH domain utilizes the classic lipid binding site for membrane association, because mutating either of two basic residues in the “signature motif” destroys membrane localization. Mutation of each basic residue of the Myo1G Myo1PH domain reveals another critical basic residue in the β3 strand, which is shared only by Myo1D. Myo1G differs from Myo1C in its phosphatidylinositol 4,5-bisphosphate dependence for membrane association, because membrane localization of phosphoinositide 5-phosphatase releases Myo1C from the membrane but not Myo1G. Thus Myo1PH domains likely play universal roles in myosin I membrane association, but different isoforms have diverged in their binding specificity.     

A Rictor-Myo1c complex participates in dynamic cortical actin events in 3T3-L1 adipocytes

Insulin signaling through phosphatidylinositol 3-kinase (PI 3-kinase) activates the protein kinase Akt through phosphorylation of its threonine 308 and serine 473 residues by the PDK1 protein kinase and the Rictor-mammalian target of rapamycin complex (mTORC2), respectively. Remarkably, we show here that the Rictor protein is also present in cultured adipocytes in complexes containing Myo1c, a molecular motor that promotes cortical actin remodeling. Interestingly, the Rictor-Myo1c complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Furthermore, while RNA interference-directed silencing of Rictor results in the expected attenuation of Akt phosphorylation at serine 473, depletion of Myo1c is without effect. In contrast, loss of either Rictor or Myo1c inhibits phosphorylation of the actin filament regulatory protein paxillin at tyrosine 118. Furthermore, Myo1c-induced membrane ruffling of 3T3-L1 adipocytes is also compromised following Rictor knockdown. Interestingly, neither the mTORC2 inhibitor rapamycin nor the PI 3-kinase inhibitor wortmannin affects paxillin tyrosine 118 phosphorylation. Taken together, our findings suggest that the Rictor-Myo1c complex is distinct from mTORC2 and that Myo1c, in conjunction with Rictor, participates in cortical actin remodeling events.     

Myo1c facilitates G-actin transport to the leading edge of migrating endothelial cells

Addition of actin monomer (G-actin) to growing actin filaments (F-actin) at the leading edge generates force for cell locomotion. The polymerization reaction and its regulation have been studied in depth. However, the mechanism responsible for transport of G-actin substrate to the cell front is largely unknown; random diffusion, facilitated transport via myosin II contraction, local synthesis as a result of messenger ribonucleic acid localization, or F-actin turnover all might contribute. By tracking a photoactivatable, nonpolymerizable actin mutant, we show vectorial transport of G-actin in live migrating endothelial cells (ECs). Mass spectrometric analysis identified Myo1c, an unconventional F-actin-binding motor protein, as a major G-actin-interacting protein. The cargo-binding tail domain of Myo1c interacted with G-actin, and the motor domain was required for the transport. Local microinjection of Myo1c promoted G-actin accumulation and plasma membrane ruffling, and Myo1c knockdown confirmed its contribution to G-actin delivery to the leading edge and for cell motility. In addition, there is no obvious requirement for myosin II contractile-based transport of G-actin in ECs. Thus, Myo1c-facilitated G-actin transport might be a critical node for control of cell polarity and motility.     

Myo1c regulates glucose uptake in mouse skeletal muscle

Contraction and insulin promote glucose uptake in skeletal muscle through GLUT4 translocation to cell surface membranes. Although the signaling mechanisms leading to GLUT4 translocation have been extensively studied in muscle, the cellular transport machinery is poorly understood. Myo1c is an actin-based motor protein implicated in GLUT4 translocation in adipocytes; however, the expression profile and role of Myo1c in skeletal muscle have not been investigated. Myo1c protein abundance was higher in more oxidative skeletal muscles and heart. Voluntary wheel exercise (4 weeks, 8.2 ± 0.8 km/day), which increased the oxidative profile of the triceps muscle, significantly increased Myo1c protein levels by ～2-fold versus sedentary controls. In contrast, high fat feeding (9 weeks, 60% fat) significantly reduced Myo1c by 17% in tibialis anterior muscle. To study Myo1c regulation of glucose uptake, we expressed wild-type Myo1c or Myo1c mutated at the ATPase catalytic site (K111A-Myo1c) in mouse tibialis anterior muscles in vivo and assessed glucose uptake in vivo in the basal state, in response to 15 min of in situ contraction, and 15 min following maximal insulin injection (16.6 units/kg of body weight). Expression of wild-type Myo1c or K111A-Myo1c had no effect on basal glucose uptake. However, expression of wild-type Myo1c significantly increased contraction- and insulin-stimulated glucose uptake, whereas expression of K111A-Myo1c decreased both contraction-stimulated and insulin-stimulated glucose uptake. Neither wild-type nor K111A-Myo1c expression altered GLUT4 expression, and neither affected contraction- or insulin-stimulated signaling proteins. Myo1c is a novel mediator of both insulin-stimulated and contraction-stimulated glucose uptake in skeletal muscle.     

Crystal Structure of Human Myosin 1c—The Motor in GLUT4 Exocytosis: Implications for Ca Regulation and 14-3-3 Binding

Myosin 1c (Myo1c) plays a key role in supporting motile events that underlie cell migration, vesicle trafficking, insulin-stimulated glucose uptake and hearing. Here, we present the crystal structure of the human Myo1c motor in complex with its light chain calmodulin. Our structure reveals tight interactions of the motor domain with calmodulin bound to the first IQ motif in the neck region. Several of the calmodulin residues contributing to this interaction are also involved in Ca^2 + binding. Contact residues in the motor domain are linked to the central β-sheet and the HO helix, suggesting a mechanism for communicating changes in Ca^2 + binding in the neck region to the actin and nucleotide binding regions of the motor domain. The structural context and the chemical environment of Myo1c mutations that are involved in sensorineural hearing loss in humans are described and their impact on motor function is discussed. We show that a construct consisting of the motor domain of Myo1c and the first IQ motif is sufficient to establish a tight interaction with 14-3-3β (K_D = 0.9 μM) and present the model of a double-headed Myo1c–14-3-3 complex. This complex has been implicated in the exocytosis of glucose transporter 4 storage vesicles during insulin-stimulated glucose uptake.     

Myosin Vc Is a Molecular Motor That Functions in Secretory Granule Trafficking

Class V myosins are actin-based motor proteins that have critical functions in organelle trafficking. Of the three class V myosins expressed in mammals, relatively little is known about Myo5c except that it is abundant in exocrine tissues. Here we use MCF-7 cells to identify the organelles that Myo5c associates with, image the dynamics of Myo5c in living cells, and test the functions of Myo5c. Endogenous Myo5c localizes to two distinct compartments: small puncta and slender tubules. Myo5c often exhibits a highly polarized distribution toward the leading edge in migrating cells and is clearly distinct from the Myo5a or Myo5b compartments. Imaging with GFP-Myo5c reveals that Myo5c puncta move slowly (∼30 nm/s) and microtubule independently, whereas tubules move rapidly (∼440 nm/s) and microtubule dependently. Myo5c puncta colocalize with secretory granule markers such as chromogranin A and Rab27b, whereas Myo5c tubules are labeled by Rab8a. TIRF imaging indicates that the granules can be triggered to undergo secretion. To test if Myo5c functions in granule trafficking, we used the Myo5c tail as a dominant negative and found that it dramatically perturbs the distribution of granule markers. These results provide the first live-cell imaging of Myo5c and indicate that Myo5c functions in secretory granule trafficking.     

Localization of Myosin 1b to Actin Protrusions Requires Phosphoinositide Binding

Myosin 1b (Myo1b), a class I myosin, is a widely expressed, single-headed, actin-associated molecular motor. Transient kinetic and single-molecule studies indicate that it is kinetically slow and responds to tension. Localization and subcellular fractionation studies indicate that Myo1b associates with the plasma membrane and certain subcellular organelles such as endosomes and lysosomes. Whether Myo1b directly associates with membranes is unknown. We demonstrate here that full-length rat Myo1b binds specifically and with high affinity to phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphatidylinositol 3,4,5-triphosphate (PIP₃), two phosphoinositides that play important roles in cell signaling. Binding is not Ca²⁺-dependent and does not involve the calmodulin-binding IQ region in the neck domain of Myo1b. Furthermore, the binding site is contained entirely within the C-terminal tail region, which contains a putative pleckstrin homology domain. Single mutations in the putative pleckstrin homology domain abolish binding of the tail domain of Myo1b to PIP₂ and PIP₃ in vitro. These same mutations alter the distribution of Myc-tagged Myo1b at membrane protrusions in HeLa cells where PIP₂ localizes. In addition, we found that motor activity is required for Myo1b localization in filopodia. These results suggest that binding of Myo1b to phosphoinositides plays an important role in vivo by regulating localization to actin-enriched membrane projections.     

Mammalian class I myosin, Myo1b, is monomeric and cross-links actin filaments as determined by hydrodynamic studies and electron microscopy

The class I myosin, Myo1b, is a calmodulin- and actin-associated molecular motor widely expressed in mammalian tissues. Analytical ultracentrifugation studies indicate that Myo1b purified from rat liver has a Stokes radius of 6.7 nm and a sedimentation coefficient, s(20,w), of 7.0 S with a predicted molar mass of 213 kg/mol. These results indicate that Myo1b is monomeric and consists primarily of a splice variant having five associated calmodulins. Molecular modeling based on the analytical ultracentrifugation studies are supported by electron microscopy studies that depict Myo1b as a single-headed, tadpole-shaped molecule with outer dimensions of 27.9 x 4.0 nm. Above a certain Myo1b/actin ratio, Myo1b bundles actin filaments presumably by virtue of a second actin-binding site. These studies provide new information regarding the oligomeric state and morphology of Myo1b and support a model in which Myo1b cross-links actin through a cryptic actin-binding site.     

Myosin Ia is required for CFTR brush border membrane trafficking and ion transport in the mouse small intestine

In enterocytes of the small intestine, endocytic trafficking of CFTR channels from the brush border membrane (BBM) to the subapical endosomes requires the minus-end motor, myosin VI (Myo6). The subapical localization of Myo6 is dependent on myosin Ia (Myo1a) the major plus-end motor associated with the BBM, suggestive of functional synergy between these two motors. In villus enterocytes of the Myo1a KO mouse small intestine, CFTR accumulated in syntaxin-3 positive subapical endosomes, redistributed to the basolateral domain and was absent from the BBM. In colon, where villi are absent and Myo1a expression is low, CFTR exhibited normal localization to the BBM in the Myo1a KO similar to WT. cAMP-stimulated CFTR anion transport in the small intestine was reduced by 58% in the KO, while anion transport in the colon was comparable to WT. Co-immunoprecipitation confirmed the association of CFTR with Myo1a. These data indicate that Myo1a is an important regulator of CFTR traffic and anion transport in the BBM of villus enterocytes and suggest that Myo1a may power apical CFTR movement into the BBM from subapical endosomes. Alternatively, it may anchor CFTR channels in the BBM of villus enterocytes as was proposed for Myo1a's role in BBM localization of sucrase-isomaltase.     

Fast adaptation in vestibular hair cells requires myosin-1c activity

In sensory hair cells of the inner ear, mechanical amplification of small stimuli requires fast adaptation, the rapid closing of mechanically activated transduction channels. In frog and mouse vestibular hair cells, we found that the rate of fast adaptation depends on both channel opening and stimulus size and that it is modeled well as a release of a mechanical element in series with the transduction apparatus. To determine whether myosin-1c molecules of the adaptation motor are responsible for the release, we introduced the Y61G mutation into the Myo1c locus and generated mice homozygous for this sensitized allele. Measuring transduction and adaptation in the presence of NMB-ADP, an allele-specific inhibitor, we found that the inhibitor not only blocked slow adaptation, as demonstrated previously in transgenic mice, but also inhibited fast adaptation. These results suggest that mechanical activity of myosin-1c is required for fast adaptation in vestibular hair cells.