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© The Rockefeller University Press, 0021-9525/1997//1287 $5.00
The Journal of Cell Biology, Volume 136, Number 6, , 1997 1287-1305

Article

Identification of Novel Graded Polarity Actin Filament Bundles in Locomoting Heart Fibroblasts: Implications for the Generation of Motile Force



Louise P. Cramer*, Margaret Siebert{ddagger}, and Timothy J. Mitchison{ddagger}

* The Randall Institute, Kings College London, University of London, London WC2B 5RL, United Kingdom; and {ddagger} Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-0450

We have determined the structural organization and dynamic behavior of actin filaments in entire primary locomoting heart fibroblasts by S1 decoration, serial section EM, and photoactivation of fluorescence. As expected, actin filaments in the lamellipodium of these cells have uniform polarity with barbed ends facing forward. In the lamella, cell body, and tail there are two observable types of actin filament organization. A less abundant type is located on the inner surface of the plasma membrane and is composed of short, overlapping actin bundles (0.25–2.5 µm) that repeatedly alternate in polarity from uniform barbed ends forward to uniform pointed ends forward. This type of organization is similar to the organization we show for actin filament bundles (stress fibers) in nonlocomoting cells (PtK2 cells) and to the known organization of muscle sarcomeres. The more abundant type of actin filament organization in locomoting heart fibroblasts is mostly ventrally located and is composed of long, overlapping bundles (average 13 µm, but can reach up to about 30 µm) which span the length of the cell. This more abundant type has a novel graded polarity organization. In each actin bundle, polarity gradually changes along the length of the bundle. Actual actin filament polarity at any given point in the bundle is determined by position in the cell; the closer to the front of the cell the more barbed ends of actin filaments face forward.

By photoactivation marking in locomoting heart fibroblasts, as expected in the lamellipodium, actin filaments flow rearward with respect to substrate. In the lamella, all marked and observed actin filaments remain stationary with respect to substrate as the fibroblast locomotes. In the cell body of locomoting fibroblasts there are two dynamic populations of actin filaments: one remains stationary and the other moves forward with respect to substrate at the rate of the cell body.

This is the first time that the structural organization and dynamics of actin filaments have been determined in an entire locomoting cell. The organization, dynamics, and relative abundance of graded polarity actin filament bundles have important implications for the generation of motile force during primary heart fibroblast locomotion.

We thank: Marc Symons, Matthew Welch, and the reviewers for thoughtfully reading the manuscript and for excellent suggestions, and also Marc for encouragement thoughout the course of this work; Aneil Mallavarapu for help on the cooled-charged coupled device microscope system and for good ideas to present Fig. 1, e and f; Kathy Franks and Roger Cooke for providing us with excellent S1 myosin preparations; Anthony Bretscher for kindly sending us anti-fimbrin antibodies, and Arshad Desai for instruction on the Nikon microscope.

This study was funded by grant GM48027 from the National Institutes of Health and a fellowship from the Packard Foundation to T.J. Mitchison, and a senior postdoctoral fellowship from the American Cancer Society, California Division to L.P. Cramer.

1. Abbreviation used in this paper: PtK2, potoroo tridactylis kidney.

Please address all correspondence to L.P. Cramer, The Randall Institute, Kings College London, University of London, 26-29 Drury Lane, London WC2B 5RL, UK. Tel.: (44)171-465-5377, Fax.: (44) 171-497-9078. E-mail: louise.cramer{at}kcl.ac.uk


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