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Myosin 1c (Myo1c) is a member of the unconventional class I myosins of vertebrates, which directly link the plasma membrane with the microfilament cortical web. Although this molecular motor has been implicated in cell functions such as cytoskeleton organization, cell motility, nuclear transcription, and endocytosis, its role in hematopoietic cells is largely unknown. In this study, we show that Myo1c is abundantly expressed in murine B lymphocytes and is preferentially located at the plasma membrane, especially in peripheral processes such as microvilli. We observed that this motor concentrates at the growing membrane protrusions generated during B cell spreading and that it is actively recruited to the immune synapse. Interestingly, Myo1c was detected in lipid rafts of B cells and showed strong colocalization with MHC-II, particularly after cross-linking of these molecules. By transfection of a dominant negative form of Myo1c or specific siRNA, we also detected alterations in the spreading and Ag-presenting ability of these cells. The data suggest that Myo1c is involved in the cytoskeleton dynamics and membrane protein anchoring or sorting in B lymphocytes.

Class I myosins regulate diverse aspects of locomotion, vesicular traffic, and peripheral process architecture in amoeba and in mammalian cells. Mass spectrometric analysis of lymphocyte fractions enriched for plasma membrane/microvilli identified myosin 1G (Myo1G) as abundant and enriched from human peripheral blood T-cells and Myo1G plus Myo1C as abundant and enriched from a mouse pre-B-cell line. To understand its structure and function, we have investigated Myo1G by immunofluorescence, by immunoblot, by transfection of wt and mutant constructs and by creation of a knockout mouse. Myo1G is abundant in multiple hematopoietic lineages, but absent in most other cell types. Endogenous Myo1G is highly enriched at the plasma membrane and in microvilli/ruffles of lymphocytes (as is transfected Myo1G in Jurkat T-cells). Structure-function analysis with truncation and point mutants show that Myo1G localization at the membrane, like Myo1C, requires a conserved "PH-like" domain present in the tail. But unlike Myo1C, it also depends on presence of its actin-binding motor domain. Knockout mice have no obvious defect in hematopoietic cell development. However preliminary studies shown that there are defects in activation of splenic T cells after stimulation with anti TCR. Studies are in progress to elucidate the underlying mechanism by which Myo1G contributes to T-cell activation.

Cell cytoskeleton plasticity is now recognized as an important element of cell to cell and cell to antigen interactions. It has been shown that T cells and dendritic cells use shape changes in order to establish effective contacts between them. B lymphocytes also show cytoskeleton rearrangements during antigen recognition. The B cell surface change upon activation with LPS or anti-CD40 plus IL-4; these stimuli induce the generation of long thin microvilli, longer than those observed in resting conditions. These structures are formed by a central actin filaments bundle with bridging associated proteins that links them to plasma membrane. One of these proteins are class I myosins, which are molecular motors that interacts both with actin fibers and plasma membrane phosphoinositides; Myo1c and Myo1g are importantly expressed in B cells and appears to increase upon activation. By using live-cell microscopy we determined that they are present across microvilli structures and seems to be involved in their formation. Finally, our data suggest a role for microvilli by segregating molecules such as MHC-II, LFA-1, CD40, etc. possibly mediated by class I myosins. Supported by CONACyT, project 56836