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 β1 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 macrophage scavenger receptor is a trimeric membrane glycoprotein with unusual ligand-binding properties which has been implicated in the development of atherosclerosis. The trimeric structure of the bovine type I scavenger receptor, deduced by complementary DNA cloning, contains three extracellular C-terminal cysteine-rich domains connected to the transmembrane domain by a long fibrous stalk. This stalk structure, composed of an alpha-helical coiled coil and a collagen-like triple helix, has not previously been observed in an integral membrane protein.

The NMR structure of an autonomously folding subdomain from villin headpiece is reported. It forms a novel three helix structure with the actin-binding residues arrayed on the C-terminal helix.

The nucleation and growth of solids from solutions impacts many natural processes and is fundamental to applications in materials engineering and medicine. For a crystalline solid, the nucleus is a nanoscale cluster of ordered atoms that forms through mechanisms still poorly understood. In particular, it is unclear whether a nucleus forms spontaneously from solution via a single- or multiple-step process. Here, using in situ electron microscopy, we show how gold and silver nanocrystals nucleate from supersaturated aqueous solutions in three distinct steps: spinodal decomposition into solute-rich and solute-poor liquid phases, nucleation of amorphous nanoclusters within the metal-rich liquid phase, followed by crystallization of these amorphous clusters. Our ab initio calculations on gold nucleation suggest that these steps might be associated with strong gold-gold atom coupling and water-mediated metastable gold complexes. The understanding of intermediate steps in nuclei formation has important implications for the formation and growth of both crystalline and amorphous materials.

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The cellular mechanism underlying the generation of -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 P-amyloid and the 3-kDa species is a heterogeneous set of peptides truncated at the ,B-amyloid N terminus. The /3-amyloid peptides are secreted in parallel with amyloid precursor protein. Inhibitors of Golgi processing inhibit secretion of P-amyloid peptides, whereas lysosomal inhibitors have no effect. The secretion of ,B-amyloid-related peptides occurs in a wide variety of cell tpes, but which peptides are produced and their absolute levels are dependent on cell type. Human astrocytes generated higher levels of (3-amyloid than any other cell type examined.These results suggest that ,B-amyloid is generated in the secretory pathway and provide evidence that glial cells are a major source of 3-amyloid production in the brain.The f3-amyloid protein is a cleavage product of the amyloid precursor protein (APP) that accumulates at high levels in the brain in Alzheimer disease, Down syndrome, and some normal elderly persons (1-3). Two major pathways of APP processing have been identified. Normal processing of APP in the secretory pathway occurs by a proteolytic cleavage within the J-amyloid sequence generating a large secreted form of the protein (4) and a smaller membrane-associated C-terminal fragment (5,6). A second pathway of APP metabolism has been identified in the endosomal-lysosomal system resulting in larger potentially amyloidogenic C-terminal fragments of APP (7-9).Recent reports have shown that f-amyloid-related peptides are normally secreted by cultured cells and can be detected in human cerebrospinal fluid (10-12). However, the cellular mechanism of this processing event is unknown.Here we show that soluble f-amyloid peptides are generated in the APP secretory pathway rather than from the degradation of APP in lysosomes. The 2) in the presence of protease inhibitors [leupeptin (5 ,ug/ml)/aprotinin (10 ,g/ml)/antipain (50 ,ug/ml)/pepstatin (5 ,ug/ml)/phenylmethylsulfonyl fluoride (100 ug/ml)] and phosphatase inhibitors (50 mM imidazole/50 mM potassium fluoride/25 mM ,B-glycerophosphate/100 ,uM sodium orthovanadate). Supernatants and lysates of transfected cells were clarified by centrifugation at 3000 x g for 20 min and 100,000 x g for 60 min, respectively, and then incubated with primary antibody for 4 hr at 4°C followed by immunoprecipitation with protein A-Sepharose (Pharmacia) for 12 hr at 4°C. The Sepharose beads were washed six times, resuspended in 2x reducing SDS sample buffer, and boiled for 3 min. The immunoprecipitated proteins were analyzed by SDS/PAGE on a 10% gel or by Tris-Tricine SDS/PAGE on a 10-20% gel (14)

Characterization of the properties of complex biomaterials using microrheological techniques has the promise of providing fundamental insights into their biomechanical functions; however, precise interpretations of such measurements are hindered by inadequate characterization of the interactions between tracers and the networks they probe. We here show that colloid surface chemistry can profoundly affect multiple particle tracking measurements of networks of fibrin, entangled F-actin solutions, and networks of cross-linked F-actin. We present a simple protocol to render the surface of colloidal probe particles protein-resistant by grafting short amine-terminated methoxy-poly(ethylene glycol) to the surface of carboxylated microspheres. We demonstrate that these poly(ethylene glycol)-coated tracers adsorb significantly less protein than particles coated with bovine serum albumin or unmodified probe particles. We establish that varying particle surface chemistry selectively tunes the sensitivity of the particles to different physical properties of their microenvironments. Specifically, particles that are weakly bound to a heterogeneous network are sensitive to changes in network stiffness, whereas protein-resistant tracers measure changes in the viscosity of the fluid and in the network microstructure. We demonstrate experimentally that two-particle microrheology analysis significantly reduces differences arising from tracer surface chemistry, indicating that modifications of network properties near the particle do not introduce large-scale heterogeneities. Our results establish that controlling colloid-protein interactions is crucial to the successful application of multiple particle tracking techniques to reconstituted protein networks, cytoplasm, and cells.