Biomedical subjects
E Sackman
Publications and source records attributed to E Sackman.
ATP-dependent membrane assembly of F-actin facilitates membrane fusion.
We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.
A precision method to measure average viscoelastic parameters of erythrocyte populations.
Suspensions of erythrocytes in media of low conductivity are subjected to homogenous high frequency electric fields (1 MHz, approximately 10 to 40 kV/m). The resulting transient deformation of the cells is measured by laser light diffraction. Employing a viscoelastic model of the erythrocyte membrane, relative values of membrane shear modulus and response time can currently be determined to within 7% or better. With a measurement time of one minute the average values of some 10(5) cells can be obtained. As a test of the method, osmotic swelling and deflation of the cells and crosslinking their membrane skeleton by diamide are used to alter the viscoelastic properties of the erythrocytes.