Microfilament dynamics during cell movement and chemotaxis monitored using a GFP–actin fusion protein

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• 作者：Monika Westphal ; Andreas Jungbluth ; Manfred Heidecker ; Bettina Mü ; hlbauer ; Christina Heizer ; Jean-Marc Schwartz ; Gerard Marriott ; Gü ; nther Gerisch
• 关键词：ng>

  BJ Berne and JE Straub ; Novel methods of sampling phase space in the simulation of biological systems ; Curr Opin Struct Biol 7 (1997) ; pp. 181– ; 189 97250755. ; //www.sciencedirect.com

• 刊名：Current Biology
• 出版年：1997
• 出版时间：March 1997
• 年：1997
• 卷：7
• 期：3
• 页码：176-183
• 全文大小：350 K

文摘

Background: The microfilament system in the cortex of highly motile cells, such as neutrophils and cells of the eukaryotic microorganism Dictyostelium discoideum, is subject to rapid re-organization, both spontaneously and in response to external signals. In particular, actin polymerization induced by a gradient of chemoattractant leads to local accumulation of filamentous actin and protrusion of a ‘leading edge’ of the cell in the direction of the gradient. In order to study the dynamics of actin in these processes, actin was tagged at its amino terminus with green fluorescent protein (GFP) and observed with fluorescence microscopy in living cells of D. discoideum.

Results: Purified GFP–actin was capable of copolymerizing with actin. In the transfected cells of D. discoideum studied, GFP–actin made up 10–20 % of the total actin. Microfilaments containing GFP–actin were capable of generating force with myosin in an in vitro assay. Observations of single living cells using fluorescence microscopy showed that the fusion protein was enriched in cell projections, including filopodia and leading edges, and that the fusion protein reflected the dynamics of the microfilament system in cells that were freely moving, being chemotactically stimulated, or aggregated. When confocal sections of fixed cells containing GFP–actin were labeled with fluorescent phalloidin, which binds only to filamentous actin, there was a correlation between the areas of GFP–actin and phalloidin fluorescence, but there were distinct sites in which GFP–actin was more prominent.

Conclusions: Double labeling with GFP–actin and other probes provides an indication of the various states of actin in motile cells. A major portion of the actin assemblies visualized using GFP–actin are networks or bundles of filamentous actin. Other clusters of GFP–actin might represent stores of monomeric actin in the form of complexes with actin-sequestering proteins.