Search

Myosin-5B is one of three members of the myosin-5 family of actin-based molecular motors. Despite its fundamental role in recycling endosome trafficking and in collective actin network dynamics, the molecular mechanisms underlying its motility are inherently unknown. Here we combine single-molecule imaging and high-speed laser tweezers to dissect the mechanoenzymatic properties of myosin-5B. We show that a single myosin-5B moves processively in 36-nm steps, stalls at ~2 pN resistive forces, and reverses its directionality at forces >2 pN. Interestingly, myosin-5B mechanosensitivity differs from that of myosin-5A, while it is strikingly similar to kinesin-1. In particular, myosin-5B run length is markedly and asymmetrically sensitive to force, a property that might be central to motor ensemble coordination. Furthermore, we show that Ca 2+ does not affect the enzymatic activity of the motor unit, but abolishes myosin-5B processivity through calmodulin dissociation, providing important insights into the regulation of postsynaptic cargoes trafficking in neuronal cells.

Rationale: Kv1.5 (KCNA5) mediates the ultra-rapid delayed rectifier current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation, Kv1.5 has emerged as a promising target for the treatment of atrial fibrillation. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known., Objective: To investigate the role of the unconventional myosin-V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5., Methods and Results: Western blot analysis showed MYO5A and MYO5B expression in the heart, whereas disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and short hairpin RNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and connexin-43 but not potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, whereas MYO5B regulated postendocytic recycling. Overexpression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling., Conclusions: MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current and, consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.

Actin-based organelle movements are driven by the related multifunctional myosin motors of class V in animals and fungi and class XI in plants. The versatility of these motors depends critically on their C-terminal globular tail domain that allows them to bind to a broad variety of cargo molecules. Regulation of this motor-cargo attachment is frequently employed to modulate organelle movement. While the overall structure of the cargo-binding globular tail appears to be conserved between myosin V and XI, it has become apparent that the motor-cargo interactions differ widely even within a single organism and involve protein complexes with different architecture and completely unrelated protein domains. At the same time, indirect evidence suggests that adaptor or receptor dimerization could facilitate efficient myosin capture. Comparison of myosin V and XI across the large evolutionary distance between animals and plants will likely reveal more fundamental insights into these important motors.

An essential feature of dendritic cell immune surveillance is endocytic sampling of the environment for non-self antigens primarily via macropinocytosis and phagocytosis. The role of several members of the myosin family of actin based molecular motors in dendritic cell endocytosis and endocytic vesicle movement was assessed through analysis of dendritic cells derived from mice with functionally null myosin mutations. These include the dilute (myosin Va), Snell's waltzer (myosin VI) and shaker-1 (myosin VIIa) mouse lines. Non muscle myosin II function was assessed by treatment with the inhibitor, blebbistatin. Flow cytometric analysis of dextran uptake by dendritic cells revealed that macropinocytosis was enhanced in Snell's waltzer dendritic cells while shaker-1 and blebbistatin-treated cells were comparable to controls. Comparison of fluid phase uptake using pH insensitive versus pH sensitive fluorescent dextrans revealed that in dilute cells rates of uptake were normal but endosomal acidification was accelerated. Phagocytosis, as quantified by uptake of E. coli, was normal in dilute while dendritic cells from Snell's waltzer, shaker-1 and blebbistatin treated cells exhibited decreased uptake. Microtubule mediated movements of dextran-or transferrin-tagged endocytic vesicles were significantly faster in dendritic cells lacking myosin Va. Loss of myosin II, VI or VIIa function had no significant effects on rates of endocytic vesicle movement., ((c) 2007 Wiley-Liss, Inc.)

The actin network has been implicated in the intracellular transport and positioning of the melanosomes, organelles that are specialized in the biosynthesis and the storage of melanin. It contributes also to molecular mechanisms that underlie the intracellular membrane dynamics and thereby can control the biogenesis of melanosomes. Two mechanisms for actin-based movements have been identified: one is dependent on the motors associated to actin namely the myosins; the other is dependent on actin polymerization. This review will focus on to the role of the actin cytoskeleton and myosins in the transport and in the biogenesis of melanosomes. Myosins involved in membrane traffic are largely seen as transporters of organelles or membrane vesicles containing cargos along the actin networks. Yet increasing evidence suggests that some of the myosins contribute to the dynamics of internal membrane by using other mechanisms. The role of the myosins and the different molecular mechanisms by which they contribute or may contribute to the distribution, the movement and the biogenesis of the melanosomes in epidermal melanocytes and retinal pigmented epithelial (RPE) cells will be discussed.

Arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Its binding to Gs-coupled vasopressin V2 receptors increases cyclic AMP (cAMP) and subsequently elicits the redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane (AQP2 shuttle), thereby facilitating water reabsorption from primary urine. The AQP2 shuttle is a paradigm for cAMP-dependent exocytic processes. Using sections of rat kidney, the AQP2-expressing cell line CD8, and primary principal cells, we studied the role of the motor protein myosin Vb, its vesicular receptor Rab11, and the myosin Vb- and Rab11-binding protein Rab11-FIP2 in the AQP2 shuttle. Myosin Vb colocalized with AQP2 intracellularly in resting and at the plasma membrane in AVP-treated cells. Rab11 was found on AQP2-bearing vesicles. A dominant-negative myosin Vb tail construct and Rab11-FIP2 lacking the C2 domain (Rab11-FIP2-DeltaC2), which disrupt recycling, caused condensation of AQP2 in a Rab11-positive compartment and abolished the AQP2 shuttle. This effect was dependent on binding of myosin Vb tail and Rab11-FIP2-DeltaC2 to Rab11. In summary, we identified myosin Vb as a motor protein involved in AQP2 recycling and show that myosin Vb- and Rab11-FIP2-dependent recycling of AQP2 is an integral part of the AQP2 shuttle.

Melanocytes and cells of the immune system share an unusual secretory mechanism which uses the lysosome as a regulated secretory organelle. Recently, a number of the proteins required for these 'secretory lysosomes' to undergo exocytosis have been identified. These include Rab27a, Lyst, Rab geranyl geranyl transferase and the adapter protein complex AP-3. Patients lacking any of these proteins are characterized by the rare combination of albinism and immunodeficiency, revealing roles for these proteins in both melanocyte and immune cell secretion. In order to ask how far the link between albinism and immunodeficiency extends we have examined cytotoxic T-lymphocyte (CTL) secretion from two BLOC-3-deficient patients and seven different mouse models of Hermansky-Pudlak syndrome, all of which display defects in pigmentation and platelet function. We find that CTL function is normal in HPS patients and pale-ear mice deficient in BLOC-3, pallid, muted and sandy mice deficient in BLOC-1, ruby-eye mice deficient in BLOC-2 and buff mice deficient in Vps33a. Similarly, the unconventional myosins, Va, VIIa and XV, which can act as effectors for Rab27a in some cell types, are not required in CTL. These results reveal differences in the protein machinery required for biogenesis and/or secretion of lysosome-related organelles in CTL and melanocytes.

The atomic force microscope (AFM) is a powerful tool for imaging biological molecules on a substrate, in solution. However, there is no effective time axis with AFM; commercially available AFMs require minutes to capture an image, but many interesting biological processes occur at much higher rate. Hence, what we can observe using the AFM is limited to stationary molecules, or those moving very slowly. We sought to increase markedly the scan speed of the AFM, so that in the future it can be used to study the dynamic behaviour of biomolecules. For this purpose, we have developed various devices optimised for high-speed scanning. Combining these devices has produced an AFM that can capture a 100 x 100 pixel image within 80 ms, thus generating a movie consisting of many successive images of a sample in aqueous solution. This is demonstrated by imaging myosin V molecules moving on mica, in solution.