ABSTRACT
Mouse embryonic fibroblasts explore the chemical suitability before spreading on a given substrate. We find this early phase of cell spreading to be characterized by transient adhesion patches with a typical mean size of (1.0 ± 0.4) µm and a lifetime of (33 ± 12) s. Eventually, these patches fuse to initiate extensive spreading of the cell. We monitor cell adhesion using reflection interference contrast and total internal reflection fluorescence microscopy. Digital time lapse movies are analysed employing spatio-temporal correlation functions of adhesion patterns. Correlation length and time can be scaled to obtain a master curve at the fusion point.
Subject(s)
Cell Adhesion/physiology , Cell Movement/physiology , Fibroblasts/cytology , Fibroblasts/physiology , Focal Adhesions/physiology , Focal Adhesions/ultrastructure , Animals , Cell Line , MiceABSTRACT
Atomic force microscopy was used to investigate the stability of dehydrated amyloid fibrils formed by human islet polypeptide (IAPP) and Abeta(1-42) peptides. IAPP amyloid fibrils were imaged in liquid (hydrated state) and in air (dehydrated). In addition, fibrils dried on the mica surface were rehydrated and re-examined both in liquid and in air (after consecutive redrying). As reported previously, the initial drying process does not result in any major change in the amyloid appearance and the dimensions of the fibrils are preserved. However, when once-dried samples are rehydrated, fibril stability is lost. The fibrils disintegrate into small particles that are attached to the mica surface. This process is further confirmed by studies of the rehydrated samples after drying, on which the morphology of the fibrils is clearly changed. Similar behavior is observed for Abeta(1-42) amyloid fibrils, which are apparently stable on first drying, but disintegrate on rehydration. The observed change indicates that dehydration is causing a change in the internal structure of the amyloid fibrils. This has important implications for studies of amyloid fibrils by other techniques. Due to the potential influence of hydration and sample history on amyloid structure, preparation and study of amyloid samples with controlled humidity requires more consideration.
Subject(s)
Amyloid/chemistry , Amyloid/ultrastructure , Desiccation/methods , Microscopy, Atomic Force/methods , Water/chemistry , Protein Conformation , Protein DenaturationABSTRACT
There has been a great deal of interest in the mechanism of lamellipodial protrusion (Pollard, T., and G. Borisy. 2003. Cell. 112:453-465). However, one of this mechanism's endpoints, the force of protrusion, has never been directly measured. We place an atomic force microscopy cantilever in the path of a migrating keratocyte. The deflection of the cantilever, which occurs over a period of approximately 10 s, provides a direct measure of the force exerted by the lamellipodial leading edge. Stall forces are consistent with approximately 100 polymerizing actin filaments per micrometer of the leading edge, each working as an elastic Brownian ratchet and generating a force of several piconewtons. However, the force-velocity curves obtained from this measurement, in which velocity drops sharply under very small loads, is not sensitive to low loading forces, and finally stalls rapidly at large loads, are not consistent with current theoretical models for the actin polymerization force. Rather, the curves indicate that the protrusive force generation is a complex multiphase process involving actin and adhesion dynamics.
