Imaging of dynamic changes of the actin cytoskeleton in microextensions of live NIH3T3 cells with a GFP fusion of the F-actin binding domain of moesin.
BACKGROUND
The cell surface undergoes continuous change during cell movement. This is characterized by transient protrusion and partial or complete retraction of microspikes, filopodia, and lamellipodia. This requires a dynamic actin cytoskeleton, moesin, components of Rho-mediated signal pathways, rearrangement of membrane constituents and the formation of focal adhesion sites. While the immunofluorescence distribution of endogenous moesin is that of a membrane-bound molecule with marked enhancement in some but not all microextensions, the C-terminal fragment of moesin co-distributes with filamentous actin consistent with its actin-binding activity. By taking advantage of this property we studied the spontaneous protrusive activity of live NIH3T3 cells, expressing a fusion of GFP and the C-terminal domain of moesin.


RESULTS
C-moesin-GFP localized to stress fibers and was enriched in actively protruding cellular regions such as filopodia or lamellipodia. This localization was reversibly affected by cytochalasin D. Multiple types of cytoskeletal rearrangements were observed that occurred independent of each other in adjacent regions of the cell surface. Assembly and disassembly of actin filaments occurred repeatedly within the same space and was correlated with either membrane protrusion and retraction, or no change in shape when microextensions were adherent.


CONCLUSIONS
Shape alone provided an inadequate criterion for distinguishing between retraction fibers and advancing, retracting or stable filopodia. Fluorescence imaging of C-moesin-GFP, however, paralleled the rapid and dynamic changes of the actin cytoskeleton in microextensions. Regional regulatory control is implicated because opposite changes occurred in close proximity and presumably independent of each other. This new and sensitive tool should be useful for investigating mechanisms of localized actin dynamics in the cell cortex.
Litman
P
P
Molecular Mechanisms of Disease Laboratories, Department of Pathology, Stanford University Medical School, 300 Pasteur Drive, Stanford, CA 94305-5324, USA. pninit@peptor.co.il
Amieva
M R
MR
Furthmayr
H
H
D016475:3T3 Cells / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D015638:Cytochalasin D / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D018715:Microscopy, Video / Q000379:methods; D017433:Protein Structure, Secondary; D011554:Pseudopodia / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012815:Signal Processing, Computer-Assisted
D000199:Actins; D003598:Cytoskeletal Proteins; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; C071058:moesin; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D