betadex and jasplakinolide

Visualization of flowing neutrophils colliding with adherent 1-mum-diameter beads presenting P-selectin allowed the simultaneous measurement of collision efficiency (epsilon), membrane tethering fraction (f), membrane tether growth dynamics, and PSGL-1/P-selectin binding lifetime. For 1391 collisions analyzed over venous wall shear rates from 25 to 200 s(-1), epsilon decreased from 0.17 to 0.004, whereas f increased from 0.15 to 0.70, and the average projected membrane tether length, L(tether)(m), increased from 0.35 mum to approximately 2.0 mum over this shear range. At all shear rates tested, adhesive collisions lacking membrane tethers had average bond lifetimes less than those observed for collisions with tethers. For adhesive collisions that failed to form membrane tethers, the regressed Bell parameters (consistent with single bond Monte Carlo simulation) were zero-stress off-rate, k(off)(0) = 0.56 s(-1) and reactive compliance, r = 0.10 nm, similar to published atomic force microscopy (AFM) measurements. For all adhesion events (+/- tethers), the bond lifetime distributions were more similar to those obtained by rolling assay and best simulated by Monte Carlo with the above Bell parameters and an average of 1.48 bonds (n = 1 bond (67%), n = 2 (22%), and n = 3-5 (11%)). For collisions at 100 s(-1), pretreatment of neutrophils with actin depolymerizing agents, latrunculin or cytochalasin D, had no effect on epsilon, but increased L(tether)(m) by 1.74- or 2.65-fold and prolonged the average tether lifetime by 1.41- or 1.65-fold, respectively. Jasplakinolide, an actin polymerizing agent known to cause blebbing, yielded results similar to the depolymerizing agents. Conversely, cholesterol-depletion with methyl-beta-cyclodextrin or formaldehyde fixation had no effect on epsilon, but reduced L(tether)(m) by 66% or 97% and reduced the average tether lifetime by 30% or 42%, respectively. The neutrophil-bead collision assay combines advantages of atomic force microscopy (small contact zone), aggregometry (discrete interactions), micropipette manipulation (tether visualization), and rolling assays (physiologic flow loading). Membrane tether growth can be enhanced or reduced pharmacologically with consequent effects on PSGL-1/P-selectin lifetimes.

Topics: Actins; beta-Cyclodextrins; Biophysics; Bridged Bicyclo Compounds, Heterocyclic; Cell Adhesion; Cell Membrane; Cell Movement; Cholesterol; Cytochalasin D; Depsipeptides; Formaldehyde; Humans; Kinetics; Mechanics; Membrane Glycoproteins; Microscopy, Atomic Force; Microspheres; Models, Statistical; Monte Carlo Method; Neutrophils; P-Selectin; Protein Binding; Selectins; Stress, Mechanical; Thiazoles; Thiazolidines

The Rho family small GTPases play a crucial role in mediating cellular responses to stretch. However, it remains unclear how force is transduced to Rho signaling pathways. We investigated the effect of stretch on the activation and caveolar localization of RhoA and Rac1 in neonatal rat cardiomyocytes. In unstretched cardiomyocytes, RhoA and Rac1 were detected in both caveolar and non-caveolar fractions as assessed using detergent-free floatation analysis. Stretching myocytes for 4 min activated RhoA and Rac1. By 15 min of stretch, RhoA and Rac1 had dissociated from caveolae, and there was decreased coprecipitation of RhoA and Rac1 with caveolin-3. To determine whether compartmentation of RhoA and Rac1 within caveolae was necessary for stretch signaling, we disrupted caveolae with methyl beta-cyclodextrin (MbetaCD). Treatment with 5 mm MbetaCD for 1 h dissociated both RhoA and Rac1 from caveolae. Under this condition, stretch failed to activate RhoA or Rac1. Stretch-induced actin cytoskeletal organization was concomitantly impaired. Interestingly the ability of stretch to activate extracellular signal-regulated kinase (ERK) was unaffected by MbetaCD treatment, but ERK translocation to the nucleus was impaired. Stretch-induced hypertrophy was also inhibited. Actin cytoskeletal disruption with cytochalasin-D also prevented stretch from increasing nuclear ERK, whereas actin polymerization with jasplakinolide restored nuclear translocation of activated ERK in the presence of MbetaCD. We suggest that activation of RhoA or Rac1, localized in a caveolar compartment, is essential for sensing externally applied force and transducing this signal to the actin cytoskeleton and ERK translocation.

Topics: Actin Cytoskeleton; Animals; Antineoplastic Agents; beta-Cyclodextrins; Caveolae; Cells, Cultured; Cyclodextrins; Cytoplasm; Depsipeptides; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Myocardial Contraction; Myocytes, Cardiac; Peptides, Cyclic; rac1 GTP-Binding Protein; Rats; Rats, Sprague-Dawley; rhoA GTP-Binding Protein; Stress, Mechanical

Recent work from our laboratory demonstrated that increased cellular cholesterol content affects the structure of the Golgi apparatus. We have now investigated the functional consequences of the cholesterol-induced vesiculation of the Golgi apparatus and the role of actin for these changes. The results showed that cholesterol-induced vesiculation and dispersion of the Golgi apparatus is a reversible process and that reversal can be inhibited by cytochalasin D, an actin-disrupting reagent. Furthermore, electron microscopy revealed that jasplakinolide, which stabilizes actin filaments, prevented the dispersion, but not the vesiculation of the Golgi cisternae. Importantly, the different Golgi markers seemed to be separated even after vesiculation. To investigate whether transport through the different steps of the exocytic pathway was affected in cholesterol-treated cells, we visualized ER to plasma membrane transport by using ts045-VSVG-GFP. In COS-1 cells expressing ts045-VSVG-GFP increased cholesterol levels did not affect transport of VSVG into the vesiculated Golgi apparatus. However, increased levels of cholesterol resulted in retention of the nascent G protein in vesicles with the TGN-marker TGN46. Biotinylation of cell surface molecules to quantify arrival of VSVG at the plasma membrane confirmed that cholesterol treatment inhibited export of the VSVG protein. In conclusion, the data show that transport of VSVG into/through a vesiculated Golgi is feasible, but that cholesterol loading inhibits exit of VSVG from the vesicles containing TGN markers. Furthermore, the data illustrate the importance of actin filaments for Golgi structure.

Topics: Actins; Animals; Antineoplastic Agents; beta-Cyclodextrins; Biological Transport; Biomarkers; Biotinylation; Cholesterol; COS Cells; Cyclodextrins; Depsipeptides; Exocytosis; Golgi Apparatus; HeLa Cells; Humans; Membrane Glycoproteins; Peptides, Cyclic; Recombinant Fusion Proteins; trans-Golgi Network; Viral Envelope Proteins