Direct Determination of Actin Polarity in the Cell

Actin filaments are polar structures that exhibit a fast growing plus end and a slow growing minus end. According to their organization in cells, in parallel or antiparallel arrays, they can serve, respectively, in protrusions or in contractions. The determination of actin filament polarity in subce...

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Bibliographic Details
Published in:Journal of molecular biology 2012-06, Vol.419 (5), p.359-368
Main Authors: Narita, Akihiro, Mueller, Jan, Urban, Edit, Vinzenz, Marlene, Small, J. Victor, Maéda, Yuichiro
Format: Article
Language:English
Subjects:
actin
Actin Cytoskeleton
Actin Cytoskeleton - chemistry
Actin Cytoskeleton - metabolism
actin filament
Animals
cell membranes
Cell Polarity
chemistry
cytoskeleton
Electron Microscope Tomography
Electron Microscope Tomography - methods
electron tomography
Fibroblasts
Fibroblasts - physiology
image analysis
metabolism
methods
microfilaments
myosin
Negative Staining
physiology
polarity of the actin filament
pseudopodia
Pseudopodia - physiology
Skin Physiological Phenomena
tomography
Trout
Trout - physiology
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Summary:Actin filaments are polar structures that exhibit a fast growing plus end and a slow growing minus end. According to their organization in cells, in parallel or antiparallel arrays, they can serve, respectively, in protrusions or in contractions. The determination of actin filament polarity in subcellular compartments is therefore required to establish their local function. Myosin binding has previously been the sole method of polarity determination. Here, we report the first direct determination of actin filament polarity in the cell without myosin binding. Negatively stained cytoskeletons of lamellipodia were analyzed by adapting electron tomography and a single particle analysis for filamentous complexes. The results of the stained cytoskeletons confirmed that all actin filament ends facing the cell membrane were the barbed ends. In general, this approach should be applicable to the analysis of actin polarity in tomograms of the actin cytoskeleton. [Display omitted] ► Actin polarity in the cell was directly determined without myosin decoration. ► Electron tomograms of negatively stained lamellipodia. ► A single particle analysis for filamentous complexes was employed.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2012.03.015