The myosin family of actin filament-based molecular motors consists of at least 20 structurally and functionally distinct classes. The human genome contains nearly 40 myosin genes, encoding 12 of these classes. Myosins have been implicated in a variety of intracellular functions, including cell migration and adhesion; intracellular transport and localization of organelles and macromolecules; signal transduction; and tumor suppression. In this review, recent insights into the remarkable diversity in the mechanochemical and functional properties associated with this family of molecular motors are discussed.

Focal segmental glomerulosclerosis is a kidney disease that is manifested as the nephrotic syndrome. It is often resistant to glucocorticoid therapy and progresses to end-stage renal disease in 50 to 70% of patients. Genetic studies have shown that familial focal segmental glomerulosclerosis is a disease of the podocytes, which are major components of the glomerular filtration barrier. However, the molecular cause in over half the cases of primary focal segmental glomerulosclerosis is unknown, and effective treatments have been elusive. We performed whole-genome linkage analysis followed by high-throughput sequencing of the positive-linkage area in a family with autosomal recessive focal segmental glomerulosclerosis (index family) and sequenced a newly discovered gene in 52 unrelated patients with focal segmental glomerulosclerosis. Immunohistochemical studies were performed on human kidney-biopsy specimens and cultured podocytes. Expression studies in vitro were performed to characterize the functional consequences of the mutations identified. We identified two mutations (A159P and Y695X) in MYO1E, which encodes a nonmuscle class I myosin, myosin 1E (Myo1E). The mutations in MYO1E segregated with focal segmental glomerulosclerosis in two independent pedigrees (the index family and Family 2). Patients were homozygous for the mutations and did not have a response to glucocorticoid therapy. Electron microscopy showed thickening and disorganization of the glomerular basement membrane. Normal expression of Myo1E was documented in control human kidney-biopsy specimens in vivo and in glomerular podocytes in vitro. Transfection studies revealed abnormal subcellular localization and function of the A159P-Myo1E mutant. The Y695X mutation causes loss of calmodulin binding and of the tail domains of Myo1E. MYO1E mutations are associated with childhood-onset, glucocorticoid-resistant focal segmental glomerulosclerosis. Our data provide evidence of a role of Myo1E in podocyte function and the consequent integrity of the glomerular filtration barrier.

GEF-H1 is a guanine nucleotide exchange factor for Rho whose activity is regulated through a cycle of microtubule binding and release. Here we identify a region in the carboxyl terminus of GEF-H1 that is important for suppression of its guanine nucleotide exchange activity by microtubules. This portion of the protein includes a coiled-coil motif, a proline-rich motif that may interact with Src homology 3 domain-containing proteins, and a potential binding site for 14-3-3 proteins. We identify GEF-H1 as a binding target and substrate for p21-activated kinase 1 (PAK1), an effector of Rac and Cdc42 GTPases, using an affinity-based screen and localize a PAK1 phosphorylation site to the inhibitory carboxylterminal region of GEF-H1. We show that phosphorylation of GEF-H1 at Ser⁸⁸⁵ by PAK1 induces 14-3-3 binding to the exchange factor and relocation of 14-3-3 to microtubules. Phosphorylation of GEF-H1 by PAK may be involved in regulation of GEF-H1 activity and may serve to coordinate Rho-, Rac-, and Cdc42-mediated signaling pathways.

Phagocytosis of invading pathogens or cellular debris requires a dramatic change in cell shape driven by actin polymerization. For antibody-covered targets, phagocytosis is thought to proceed through the sequential engagement of Fc-receptors on the phagocyte with antibodies on the target surface, leading to the extension and closure of the phagocytic cup around the target. We find that two actin-dependent molecular motors, class 1 myosins myosin 1e and myosin 1f, are specifically localized to Fc-receptor adhesions and required for efficient phagocytosis of antibody-opsonized targets. Using primary macrophages lacking both myosin 1e and myosin 1f, we find that without the actin-membrane linkage mediated by these myosins, the organization of individual adhesions is compromised, leading to excessive actin polymerization, slower adhesion turnover, and deficient phagocytic internalization. This work identifies a role for class 1 myosins in coordinated adhesion turnover during phagocytosis and supports a mechanism involving membrane-cytoskeletal crosstalk for phagocytic cup closure.