Paul Matsudaira

Small amounts (7-250 pmol) of myoglobin, beta-lactoglobulin, and other proteins and peptides can be spotted or electroblotted onto polyvinylidene difluoride (PVDF) membranes, stained with Coomassie Blue, and sequenced directly. The membranes are not chemically activated or pretreated with Polybrene before usage. The average repetitive yields and initial coupling of proteins spotted or blotted into PVDF membranes ranged between 84-98% and 30-108% respectively, and were comparable with the yields measured for proteins spotted onto Polybrene-coated glass fiber discs. The results suggest that PVDF membranes are superior supports for sequence analysis of picomole quantities of proteins purified by gel electrophoresis.

Networks of cross-linked and bundled actin filaments are ubiquitous in the cellular cytoskeleton, but their elasticity remains poorly understood. We show that these networks exhibit exceptional elastic behavior that reflects the mechanical properties of individual filaments. There are two distinct regimes of elasticity, one reflecting bending of single filaments and a second reflecting stretching of entropic fluctuations of filament length. The mechanical stiffness can vary by several decades with small changes in cross-link concentration, and can increase markedly upon application of external stress. We parameterize the full range of behavior in a state diagram and elucidate its origin with a robust model.

Cell migration on 2D surfaces is governed by a balance between counteracting tractile and adhesion forces. Although biochemical factors such as adhesion receptor and ligand concentration and binding, signaling through cell adhesion complexes, and cytoskeletal structure assembly/disassembly have been studied in detail in a 2D context, the critical biochemical and biophysical parameters that affect cell migration in 3D matrices have not been quantitatively investigated. We demonstrate that, in addition to adhesion and tractile forces, matrix stiffness is a key factor that influences cell movement in 3D. Cell migration assays in which Matrigel density, fibronectin concentration, and beta1 integrin binding are systematically varied show that at a specific Matrigel density the migration speed of DU-145 human prostate carcinoma cells is a balance between tractile and adhesion forces. However, when biochemical parameters such as matrix ligand and cell integrin receptor levels are held constant, maximal cell movement shifts to matrices exhibiting lesser stiffness. This behavior contradicts current 2D models but is predicted by a recent force-based computational model of cell movement in a 3D matrix. As expected, this 3D motility through an extracellular environment of pore size much smaller than cellular dimensions does depend on proteolytic activity as broad-spectrum matrix metalloproteinase (MMP) inhibitors limit the migration of DU-145 cells and also HT-1080 fibrosarcoma cells. Our experimental findings here represent, to our knowledge, discovery of a previously undescribed set of balances of cell and matrix properties that govern the ability of tumor cells to migration in 3D environments.

The cellular mechanism underlying the generation of beta-amyloid in Alzheimer disease and its relationship to the normal metabolism of the amyloid precursor protein are unknown. In this report, we show that 3- and 4-kDa peptides derived from amyloid precursor protein are normally secreted. Epitope mapping and radiolabel sequence analysis suggest that the 4-kDa peptide is closely related to full-length beta-amyloid and the 3-kDa species is a heterogeneous set of peptides truncated at the beta-amyloid N terminus. The beta-amyloid peptides are secreted in parallel with amyloid precursor protein. Inhibitors of Golgi processing inhibit secretion of beta-amyloid peptides, whereas lysosomal inhibitors have no effect. The secretion of beta-amyloid-related peptides occurs in a wide variety of cell types, but which peptides are produced and their absolute levels are dependent on cell type. Human astrocytes generated higher levels of beta-amyloid than any other cell type examined. These results suggest that beta-amyloid is generated in the secretory pathway and provide evidence that glial cells are a major source of beta-amyloid production in the brain.