Microtubule plus end tracking proteins or +TIPS have been implicated in the control of microtubule dynamics, cell polarity, spindle positioning, and organelle: microtubule interactions that precede dynein-dependent organelle motility. We have shown that the distribution of melanosomes in mouse melanocytes is driven by long range, bi-directional, microtubule-dependent movements and local, myosin Va-dependent movements on actin in the periphery. We have also shown that Myosin Va is recruited on to the melanosome surface by a receptor complex containing Rab27a present on the melanosome membrane and melanophilin, which links myosin Va to Rab27a. We now show that GFP-tagged melanophilin also exhibits microtubule plus end tracking behavior. Moreover, GFP-tagged myosin Va can also surf the microtubule plus end and does so in a melanophilin-dependent manner. Finally, alterations in the cellular levels of the +TIP EB1, as well as pull down assays, argue that melanophilin surfs indirectly by hitchhiking on EB1. These results indicate that vertebrate cells have retained in the form of a myosin Va-melanophilin-EB1 complex the connection between microtubule plus ends and F-actin seen in the yeast complex containing Myo2p (a type V myosin), Bim1p (an EB1 homolog), and Kar9p, which links Myo2p to Bim1p. Given melanophilin?s documented role in coupling Rab27a-bearing vesicular cargo to myosin Va, we suggest that the accumulation of melanophilin and myosin Va at the microtubule plus end may serve to focus and facilitate the transfer of melanosomes from microtubules to actin at this location. The contractile vacuole (CV) complex is a specialized intracellular membrane compartment that serves as the osmoregulatory organelle in protozoa. In Dictyostelium, this compartment is composed of an interconnected network of tubules and cisternae or bladders. These membranes accumulate excess water (e.g. rain water) that has entered the cell by osmosis by pumping protons and most likely bicarbonate into their lumen. The resulting ion gradient draws the excess water out of the cytoplasm and into the lumen. The swollen bladders that are generated expel this excess water from the cell through a transient fusion pore in the plasma membrane. The tubules and bladders that comprise the system are highly dynamic and very pleiomorphic, are rapidly interconvertible, and do not mix with the endosomal/lysosomal membrane system or with the plasma membrane during the process of water expulsion. What has emerged from prior studies is a working definition of a CV membrane cycle in Dictyostelium in which swollen, mature bladders contact the fusion pore in the plasma membrane, water is discharged from the cell, the collapsed bladder membrane folds up into a tight knot immediately under the plasma membrane, this knot of membrane rapidly transforms into tubules that radiate out across the actin rich cortex, and these tubules fuse with each other and with immature bladders during the filling phase to create new mature bladders. These cortical events are seen best in time lapse confocal images of the ventral surface of adherent cells, as this configuration places a large area of the plasma membrane and subjacent actin-rich cortex within a single focal plane. The close association of CV membranes with the actin-rich cortex, and the dramatic motility of CV tubules along the cortex have led to the suggestion that CV membranes recruit some type of myosin. We now show that the Dictyostelium type V myosin myoJ is targeted to CV membranes and is responsible for their steady state association with the actin-rich cortex. Moreover, we show that myo J drives the tubulation of collapsed bladder membranes along the cortex following water discharge. Finally, the steady state accumulation of CV membranes around the MTOC seen in myoJ null cells forced us to visualize CV membrane dynamics in the middle off the cell as well as along its ventral surface. These images revealed that the tubules emanating from collapsed bladders move not only on actin in the plane of the membrane but bidirectionally along microtubules between the cortex and the microtubule organizing center (MTOC) adjacent to the nucleus. From this we conclude that myoJ cooperates with plus and minus end-directed microtubule motors to drive the proper distribution and function of the CV complex in Dictyostelium. The coat color phenotypes of dilute (myosin Va-), ashen (Rab27a-) and leaden (melanophilin-) mice are identical and nonadditive because the absence of any one of these gene products completely abrogates the interaction of melanosomes with the actin cytoskeleton. We have begun to characterize dilute suppressor (dsu), an extragenic suppressor of dilute, ashen and leaden, which our collaborators recently identified by backcrossing and bac transgene rescue. Dsu encodes a novel, highly-charged protein of ~22 kDa. Consistent with the deletion mutation present in the dsu allele, dsu melanocytes are devoid of the dsu protein (dsup). Therefore, it is the loss of expression of dsup that causes rescue. Dilute melanocytes that are also homozygous dsu show a significant spreading of melanosomes throughout the cytoplasm, which most likely explains the restoration of coat color. But melanosomes in these cells never concentrate in dendritic tips, presumably because this process is strictly myosin Va-dependent. Consistently, overexpression of GFP-tagged dsup in dilute/dsu melanocytes causes melanosomes to once again cluster in the cell center. Moreover, these cells show an almost exclusive colocalization of GFP-tagged dsup with melanosomes. Melanosome targeting of dsup requires the addition of multiple palmitates at a cluster of six cysteines located near its N-terminus, suggesting that dsup may be a component of a specialized melanosomal membrane micro domain. We are now characterizing the nature of microtubule-based melanosome movements in melanocytes lacking and over expressing dsup to determine if dsup causes rescue by effecting the balance between plus and minus end-directed movements of melanosomes on microtubules.
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