• Microfilament-membrane attachments in Xenopus laevis fibroblasts and myocytes.

      Samuelsson, Steven John (1992)
      Quick-freeze, deep-etch, rotary-replication electron microscopy was used to study microfilament-membrane interactions. Three main issues were examined in this work: (1) whether microfilaments associate with the membrane end-on or laterally; (2) the types of structures at focal contacts which contain vinculin, talin, {dollar}\beta\sb1{dollar}-integrin and {dollar}\alpha{dollar}-actinin; and (3) the similarities and differences between microfilament-membrane interactions in myocytes and fibroblasts. The cytoplasmic surfaces of cultured Xenopus fibroblast cells were exposed by shearing and, in some cases, labeled with antibodies to proteins known to be enriched at focal contacts. Bundles of actin microfilaments associated with fibroblast plasma membrane at focal contacts in three distinct ways. The most extensive association occurred at aggregates of irregular globules that projected into the cytoplasm. Other connections with the plasma membrane were mediated by two kinds of short filaments: one has the diameter and platinum repeat typical of microfilaments, the other is smooth and is about half this diameter. The aggregates, but not the two kinds of filaments, labeled with antibodies to vinculin, talin and {dollar}\beta\sb1{dollar}-integrin; the latter was labeled especially well when overlying structures have been removed. A smaller membrane-associated aggregate of particulate material lies between focal contacts; these are devoid of vinculin and talin but are labeled with integrin antibodies. Antibodies to {dollar}\alpha{dollar}-actinin and filamin labeled bundles of microfilaments and aggregates at focal contacts, but the frequency of labeling the aggregates was much lower than labeling by antibodies to talin and {dollar}\beta\sb1{dollar}-integrin and less than aggregates labeled for vinculin.;Focal contact-like attachments were found in myocytes, but these were less well organized than in fibroblasts. Aggregates of globular material filled the space between the microfilaments and the membrane. Antibodies to vinculin and talin specifically labeled this material. A second adhesive domain covered large areas of the myocyte membrane, in which irregular, branched microfilaments individually contacted small masses of particles on the membrane. Vinculin, talin and {dollar}\beta\sb1{dollar}-integrin antibodies labeled these masses close to the membrane. In addition, a third microfilament-membrane attachment domain was observed. This structure consisted of masses of particle-aggregates and microfilaments and labeled densely with talin and vinculin; the identity of this domain was unknown but could be analogous to a myotendinous junction. Thus, microfilaments at focal contacts and focal attachments associate laterally with the membrane at aggregates of particles enriched in vinculin, talin and {dollar}\beta\sb1{dollar}-integrin. Thin short filaments assist in anchoring microfilaments at focal contact of fibroblasts. A second, irregular adhesion anchors large areas of the myocyte cytoskeleton to the membrane; vinculin, talin and integrin are contained in this adhesive structure. Strands of extracellular filaments could also be seen at some sites associated with focal contacts and myocyte focal attachments.