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DFG - Projekt: Myosin-Based Organelle Motility
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PROJEKT: In previous studies, we showed that organelles in the giant axon of the squid switch between actin filaments and microtubules during movement (Kuznetsov et al., 1992, Nature, 356:722-725; see movie). These observations provided the first evidence that both microtubules and actin filaments are required for fast axonal transport.
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Our working hypothesis is that microtubules provide the tracks for movement over long distances while actin filaments provide the tracks for movement locally. Long distance movement is defined as movement of vesicles from one location in the cell to another when the sites are physically separated. Local movement, on the other hand, is defined as (a) movement of vesicles from a functional site to a closely adjacent microtubule domain in the cytoplasm and (b) from a microtubule domain to the actin rich cortex at the plasma membrane or, in the case of neurons, to the actin network in the dendritic spines and axon terminals. This model of transport, referred to as the dual filament model of transport, is thought to be a general mechanism of transport of vesicles in cells and may include transport of cytosolic components such as mRNA.
ER isolated from Axons is transported on Actin Filaments and contains Myosin V
We developed a procedure to isolate endoplasmic reticulum (ER) from axoplasm extruded from the giant axon of the squid. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors (see movie). An antibody to squid myosin V was used in immunogold EM studies to show that myosin V localized to these vesicles. Furthermore, we have been able to show that movement of ER vesicles on actin filaments is driven by an unconventional myosin of the type V class.
Fig. 1: Actin-dependent movement of ER on exogenous actin filaments. (A) Video microscopy images of an ER vesicle moving on rhodamine phalloidin-labeled actin filaments. Since actin filaments are smaller than the detection level of AVEC-DIC microscopy, the organelles appeared to move on invisible tracks. Asterisk denotes where the ER attached to the glass coverslip. The large vesicle moved from the site of attachment during the first 20 seconds, retracted briefly, extended forward until 32 seconds, retracted again, extended until 52 seconds and then retracted again. Note that the tubular membrane was clearly visible when the vesicle moved forward from the site of attachment. (B) Diagram of the path of the vesicle shown in A.
Fig. 2: Electron micrographs demonstrating co-localization of myosin V (small gold beads) and kinesin (large gold beads) on ER vesicles. (A-D) Axoplasmic ghosts were reacted with an antibody to squid myosin V and an antibody to squid kinesin heavy chain (Inset in panel B shows small vesicles). The labeled ghosts were incubated with 10-nm gold-labeled secondary antibody for the myosin antibody and 20-nm gold-labeled secondary antibody for the kinesin antibody. The gold-labeled ghosts were transferred to a carbon-Formvar coated grid and stained with uranyl acetate. (E) ER was stained for myosin only. (F) The primary antibodies were replaced with pre-immune sera to demonstrate the specificity of the labeling reaction. These data demonstrate that the microtubule motor, kinesin, and the actin filament motor, myosin V, are located on the same ER vesicles. The co-localization of myosin V and kinesin on the same vesicle provides the means by which a vesicle is able to switch between actin filaments and microtubules during movement. Scale bars = 100 nm.
Myosin V Mediates the Interaction of Phagosomes with Actin Filaments in Vitro and Takes a Part in Phagosome Movement in Vivo.
During the early stages of phagocytosis, the plasma membrane adjacent to the particle to be internalized becomes tightly associated with actin filaments (AFs). However the existence of an interaction between mature, "late", phagosomes and AFs after internalization remains unclear. To investigate the role of myosin Va in phagocytosis, we established a light microscopy-based assay that reconstitutes the binding of phagosomes purified from mouse macrophages to pre-assembled AFs in vitro. Experiments measuring the binding of phagosomes with AFs demonstrated that both endogenous myosin Va from mouse macrophages and exogenous myosin Va from chicken brain stimulated the phagosome-AF interaction. Myosin Va association with phagosomes correlated with their ability to bind AFs in an ATP-dependent manner and antibodies to myosin Va specifically blocked the ATP-dependent phagosome binding to AFs. We have studied the involvement of myosin Va in the accumulation of phagosomes at the perinuclear region of macrophages. The uptake and retrograde transport of phagosomes from the periphery to the center of cells were observed in bone marrow macrophages from both normal mice and mice homozygous for the dilute-lethal spontaneous mutation (myosin Va null). However, in dilute-lethal macrophages the accumulation of phagosomes was occurred twofold faster than in normal macrophages (see movies). These observations demonstrate that myosin Va is involved in the later events of phagocytosis and mediates the interaction between phagosomes AFs resulting in delay of microtubule-dependent retrograde phagosome movement towards the cell center. Despite myosin Va binds to phagosomes, this binding is not required for phagosome retrograde movement toward cell center, but appears to be necessary for short-range transport of phagosomes at the cell periphery, phagosome maturation and fusion with endocytic organelles.
Fig. 3: The reconstitution of phagosome binding to pre-assembled actin filaments (AFs) in vitro. (A-B) The fluorescence images of typical fields of the binding assay. (A) Rhodamine-phalloidin labeled AFs were absorbed on the coverslip surface. (B) Phagosomes bound to AFs in the peresence of 1 mg/ml cytosol after washing out of unbound phagosomes were observed by fluorescence microscopy . Bars 10 µm. (C) Stimulation of phagosome binding to AFs by cytosolic factor(s). Phagosome binding to the AFs lawn of uninternalized latex beads (beads-cyt; beads+cyt). Binding in the presence and absence of 1 mg/ml cytosol of phagosomes isolated after FSG beads internalization (1 hr pulse, 1 hr chase) (ph+cyt, ph-cyt). Binding of phagosomes treated with 100 µg/ml trypsin (trypsin ph+cyt). Binding of phagosomes in the presence of 5 µM cytochalasin D (ph+cyt+CD), 15 µM DNase I (ph+cyt+DNase I) and 5 µM latrunculin (ph+cyt+Lat).
Fig. 4: The accumulation of the phagosomes in the perinuclear region of normal and dilute-lethal MBMM cells. (A-B) VEC-DIC images of the normal cells. Cells were loaded with latex beads for 15 min (A) and then chased for 4 hrs (B). The accumulation of phagosomes is seen at the cell centers (arrows). The same accumulation was observed in dilute-lethal MBMM cells (not shown). Bar 10 µm. (C) Detection of myosin Va in normal and dilute-lethal MBMM cells. SDS-PAGE of lysates of normal (N) and dilute-lethal (D) MBMM cells; immunoblot of lysates of normal (N’) and dilute-lethal (D’) MBMM cells of probed with DIL2 antibody. No myosin Va was detected in dilute-lethal MBMM cells. (D) Immunofluorescence staining of normal MBMM cells engaged in phagocytosis with DIL2 antibody against myosin Va. Fluorescent patches were found associated with some phagosomes enclosing blue latex beads in normal but not in dilute-lethal MBMM cells (arrows). Insert- 2 folds higher magnification. Bar 10 µm. (E) Kinetics of phagosome accumulation near the cell center in normal and dilute-lethal MBMM cells based on three independent experiments. Cells were loaded for 15 min, washed for 15 min and then chase for 15, 30, 45, 60 and 240 min. For each time point 20 cells were analyzed. The increase of accumulation rate was found in dilute-lethal MBMM cells (see time points 15, 30 and 45 min). (F) Kinetics of phagosome accumulation near the cell center in normal (Normal) and dilute-lethal (Dilute) MBMM cells in the presence of nocodazole. The cells were treated as in (E) and chased for 1 hr and 4 hr in media with 5 µM nocodazole (Noc).
Fig. 5: Phagosome movement in the periphery of normal and dilute-lethal MBMM cells (see movies). (A) Successive video frames (3 s intervals) of centripetal phagosome movements (arrowheads) in normal (a) and dilute-lethal (b) MBMM cells. (B) Typical paths for phagosome movements occurred over a 2min period. (1-2) The paths in the periphery of normal MBMM cells (1) and dilute-lethal MBMM cells (2). (3) The paths in the perinuclear region of normal (the 2 upper) and dilute-lethal (the 2 lower) MBMM cells.
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QuickTime Movie 1 - An individual organelle moves on both actin filaments and microtubule:
 324_EN_mt-acsq1.mov
QuickTime Movie 2 - An individual organelle moves on both actin filaments and microtubule:
325_EN_mt-acsq2.mov
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Naturwissenschaften 
Neue Medikamente
für Projekte
uni-rostock.de
