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First published online 17 August 2004
doi: 10.1242/jcs.01309

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The bi-directional translocation of MARCKS between membrane and cytosol regulates integrin-mediated muscle cell spreading

Marie-Hélène Disatnik^1,*, Stéphane C. Boutet^1,*, Wilfred Pacio¹, Annie Y. Chan¹, Lindsey B. Ross¹, Christine H. Lee¹ and Thomas A. Rando^1,2,

¹ Department of Neurology and Neurological Sciences, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5235, USA
² GRECC and Neurology Service, Veterans Affairs Palo Alto Heath Care System, 3801 Miranda Avenue, Palo Alto, CA 94304, USA

View larger version (45K): [in a new window]   Fig. 1. MARCKS expression and its phosphorylation in myoblasts. (A) Representative northern blot showing the expression of MARCKS transcript in myoblasts in culture. The expression in brain was used as a positive control. 18S ribosomal RNA was used for a loading control. (B) Western blot analysis showing the expression of MARCKS protein in myoblasts in culture. Brain protein was used as a positive control. (C) Western blot analysis showing MARCKS distribution between membrane pellet (P) and cytosolic (C) compartments at various time points after plating cells on fibronectin (FN). (D) Western blot analysis of phosphorylated MARCKS in myoblasts as a function of time after plating myoblasts on fibronectin. The expression of MARCKS protein did not show any significant changes over the same time course. (E) In vivo phosphorylation labeling of MARCKS. Myoblasts were labeled with [32P]-orthophosphate after plating on fibronectin. Phosphorylated MARCKS was immunoprecipitated at indicated times after plating and run on SDS-PAGE as described in Materials and Methods. The amount of MARCKS immunoprecipitated is shown after probing the blot with an anti-MARCKS polyclonal antibody. Studies were done in the absence or presence of the PKC inhibitor, calphostin C, as indicated. (F) Phosphorylation of MARCKS in control myoblasts (GFP vector alone) and in myoblasts overexpressing PKC-GFP. MARCKS phosphorylation is shown by western blot analysis at different time points after the cells were plated on fibronectin.

View larger version (40K): [in a new window]   Fig. 2. Localization of MARCKS fusion proteins. (A) Cells transfected with various MARCKS fusion proteins or control cells were subjected to fractionation, and the distribution of the fusion proteins between the membrane pellet (P) or cytosolic (C) fractions was assessed by western blot analysis using an antibody against DsRed. The arrow indicates DsRed protein alone at 30 kDa in control transfected cells. (B) The localization of each MARCKS-DsRed fusion protein was assessed by microscopic analysis of DsRed immunofluorescence. Myoblasts were plated on fibronectin for 30 minutes. Control cells were transfected with a vector expressing DsRed, and all other cells were transfected with MARCKS-DsRed fusion proteins, as indicated. The wild-type (WT) form was localized at the cell membrane and focal adhesion sites (arrow), G2A as well as G2A-mPSD forms were found mostly cytosolic with a perinuclear staining pattern notable in G2A-mPSD cells, and both PSD mutants showed punctate staining patterns. Bar, 5 µm. (C) Phosphorylation of MARCKS fusion proteins. Myoblasts expressing the vector alone (control), wild-type MARCKS (WT) or the G2A mutant (G2A) were plated on fibronectin for 1 hour. The level of MARCKS phosphorylation was measured by western blot analysis using an antibody against phosphorylated MARCKS. Endogenous phosphorylated MARCKS is indicated with an asterisk. (D) Western blot analysis using an anti-DsRed antibody showing the subcellular localization of MARCKS fusion proteins prepared from overexpressing cells plated on fibronectin for 1 hour and 4 hours, as indicated, followed by cellular fractionation. Wild-type and G2A MARCKS were found mainly in the cytosol (C) after 1 hour and in the membrane pellet (P) after 4 hours on fibronectin.

View larger version (64K): [in a new window]   Fig. 3. Spreading of myoblasts in the presence of mutant forms of MARCKS. (A) The spreading of cells expressing the wild type, G2A-mPSD and PSD fusion proteins was monitored after the cells were plated on fibronectin (FN) or laminin (LM) for 30 minutes. G2A-mPSD and PSD cells failed to spread on fibronectin. (B) Cells were allowed to spread on laminin (open bars) or fibronectin (filled bars) for 30 minutes as in panel (A), and cell spreading was quantified by determining the percentage of cells that had spread (see Materials and Methods). Data represent the mean±SD of three independent experiments. (C) The spreading of cells expressing the G2A fusion protein compared to control and wild-type (WT) cells after 5 or 15 minutes on fibronectin. Cells expressing the G2A mutant spread faster than both other cell types. (D) Cells overexpressing MARCKS fusion proteins were treated with PMA (100 nM) and plated on fibronectin for 30 minutes. Treatment with PMA accelerated the spreading of cells expressing wild-type or G2A fusion proteins (although at this time point the difference with or without PMA is minimal because the cells have are already spread) but failed to rescue the spreading deficit of cells expressing the G2A-mPSD and PSD mutants. Bars, 10 µm.

View larger version (65K): [in a new window]   Fig. 4. Inhibition of MARCKS dephosphorylation by phosphatase inhibitors alters cell spreading. (A) The phosphorylation of MARCKS in myoblasts plated on fibronectin in the presence or absence of the phosphatase inhibitors, okadaic acid or microcystin. MARCKS phosphorylation was analyzed in untreated cells (lane 1), in cells treated with okadaic acid at 50 nM or 100 nM (lanes 2 and 3 respectively) and in cells treated with microcystin at 2 µM or 3 µM (lanes 4 and 5 respectively) 5 hours after cells were plated on fibronectin. (B) Prior to analysis of MARCKS phosphorylation in panel (A), cells were photographed (20x) to show the inhibition of spreading in the presence of 50 nM okadaic acid and 2 µM microcystin compared to untreated cells. Bar, 10 µm.

View larger version (49K): [in a new window]   Fig. 5. Focal adhesion and actin stress fiber formation in cells expressing MARCKS mutants. (A) Focal adhesion sites were labeled with an anti-FAK antibody 2 hours after cells were plated on fibronectin. Control cells were transfected with empty vector. (B) Cells were plated on fibronectin for 2 hours, fixed, and stained with Phalloidin Alexa488 to label stress fibers. (C) Quantification of actin polymerization by sedimentation assay in cells expressing MARCKS fusion proteins. Cell extracts were analyzed by western blotting for total actin and the amount of actin in the G-actin pool or the F-actin pool. Bars, 2 µm.

View larger version (55K): [in a new window]   Fig. 6. The temporal sequence of MARCKS translocation and regulation of actin dynamics during cell adhesion and spreading. (A) Wild-type MARCKS and G2A mutant translocate from the membrane to the cytosol upon phosphorylation on the PSD domain (red circle) to induce cell spreading. Because the G2A mutant is less tightly associated with the membrane, the early stages of cell spreading are actually enhanced by the expression of this protein. The deletion (PSD) or mutation (mPSD) of the PSD domain keeps MARCKS at the membrane bound by the myristoylation domain (green cone) and cells expressing these mutants fail to spread on fibronectin. The double mutant (G2A-mPSD) is almost exclusively cytosolic because of the lack of the myristoylation domain and the weak electrostatic interaction between the mutant PSD domain (pink circle) and the membrane. As this mutant cannot cycle back to the membrane, cells expressing this mutant also fail to spread on fibronectin. (B) A model of the process of cell spreading mediated by 5ß1 integrin. The first stage (`cell attachment') begins with the binding of 5ß1 integrin to fibronectin. At this stage, PKC is in an inactive form in the cytosol and MARCKS is found at the membrane. The second stage (`cell adhesion') begins by the clustering of integrins and subsequent activation and membrane association of PKC, which phosphorylates MARCKS and mediates its translocation to the cytosol. During cell adhesion, FAK is co-localized with cortical actin at the leading edge. The two processes, `attachment' and `adhesion' are difficult to assess independently, and most studies testing the initial stages of cell spreading would involve a combination of the two. The final stage (`cell spreading') is characterized by the inactivation of PKC and the dephosphorylation of MARCKS and its translocation back to the membrane where it cross-links actin leading to the formation of stress fibers. At this stage, FAK is localized at focal adhesion sites that, along with the maturing actin cytoskeleton, maintain the cell in an adherent spread state.