Actin filament organization in aligned prefusion myoblasts - PubMed (original) (raw)
Actin filament organization in aligned prefusion myoblasts
Nathan T Swailes et al. J Anat. 2004 Nov.
Abstract
The organization of the actin cytoskeleton in prefusion aligning myoblasts is likely to be important for their shape and interaction. We investigated actin filament organization and polarity by transmission electron microscopy (TEM) in these cells. About 84% of the filaments counted were either found in a subplasmalemma sheet up to 0.5 microm thick that was aligned with the long axis of the cell, or in protrusions. The remaining filaments were found in the cytoplasm, where they were randomly orientated and not organized into bundles. The polarity of the subplasmalemma filaments changed progressively from one end of the cell to the other. At the ends of the cells and in protrusions, the majority of filaments were organized such that their barbed ends faced the tip of the protrusion. We did not find any actin filament bundles or stress fibres in these cells. Time-lapse phase microscopy demonstrated that aligned cells were still actively migrating at the time of our TEM observations, and their direction of movement was restricted to the long axis of the cell group. The ability of these cells to locomote actively in the absence of actin filament bundles suggests that in these cells the subplasmalemma actin sheet contributes not only to cell shape but also to cell locomotion.
Figures
Fig. 1
Embedding a monolayer of cells. (a) Cells, fixed and prepared for electron microscopy on their circular coverslips, were placed on squares of Aclar, and coated with a drop of Araldite. An Aclar washer was placed on top. (b) A second square of Aclar was pressed onto the upper surface of the washer to sandwich the Araldite-coated coverslip and washer. The Araldite was then polymerized at 60 °C for 24 h. (c) After polymerization the Aclar squares, washer and coverslip were peeled away and a hardened Araldite disc with the cells embedded remained. Cells were selected based on their morphology under the light microscope and the part of the disc containing them was excised using a scalpel. (d) These cells were then flipped over 180° so that their ventral surface was uppermost and mounted onto an Araldite stub using rapid cure Araldite and a specimen mounting apparatus. (e) The block was shaped and (f) 60–90-nm silver/gold sections were cut using a diamond knife and ultramicrotome.
Fig. 2
Electron micrographs of aligned myoblasts, permeabilized with saponin and decorated with S1. (a) A low-power montage of electron micrographs depicting a ventral section through several saponin permeabilized cells. (b–d) Enlargements of the corresponding areas within the montage; (e) from a control cell not shown (scale bar 250 nm). In (a) long vertical arrows highlight the regions between which the subplasmalemma filaments were measured. Long dashed arrow links two parts of a single cell separated by a protrusive structure extending from another cell, highlighting the ‘bridging’ interactions of neighbouring cells. The montage shows that some cells were mildly permeabilized (white asterisks), whilst others were not permeabilized (black asterisk) and acted as controls for morphology (scale bar 4 µm). Panel (b) shows an area of S1-labelled subplasmalemma filaments. The plasma membrane is located at the bottom of the image. Panel (c) shows S1-labelled actin filaments within the protrusive end of a cell. Panel (d) shows S1-labelled actin filaments within the cytoplasm. Panel (e) shows the subplasmalemma region of a non-permeabilized control cell. Added arrowheads in (b), (c) and (d) emphasize the orientatation of S1-labelled actin filaments. In (e) opposing arrowheads indicate the intact plasma membrane, and the long arrow highlights the location of undecorated subplasmalemma actin filaments. Panel (f) shows a phase contrast image taken of myoblasts at the same stage of development as that for the EM above, fixed using 4% paraformaldehyde and viewed in the light microscope, and (g) shows the fluorescent image of the same cells stained for filamentous actin with rhodamine phalloidin.
Fig. 3
Graphs showing the graded polarity of actin filaments in the subplasmalemma zone for the ventral sections of three cells within an aligned group. The three symbols represent data taken from each of the three ventral sections. In these sections the subplasmalemma sheet runs parallel to the plasma membrane and spans the entire length of the cell.
Fig. 4
Short-range random polarity of subplasmalemma actin filaments in a ventral section of an aligned myoblast. Transects were drawn at 90° to the membrane every 0.1 µm over a 5-µm stretch at the central region of the cell. Polarity was scored where filaments intersected these lines. To avoid bias, transects were scored in a random sequence. Points a–d are shown in the micrograph. The total number of filaments scored = 406. Although the filament polarity appears to shift from about 80% that point towards one end of cell (left-hand side of micrograph) to 20% in an alternating fashion, in fact these observations can be explained by a random organization of filaments with mixed polarity. The polarity of the filaments is indicated by the filled and empty arrows; filled arrows represent filaments pointing towards one end of the cell, and the empty arrows represent filaments pointing towards the other end.
Fig. 5
Analysis of cell locomotion within aligned groups. The cells contributing to the aligned group shown in (b) and highlighted with black asterisks (n = 26) were quantified and analysed as described in the Materials and methods section. In each case, the cell tracks are shifted to a common point of origin as shown in the trajectory diagram (a) and a horizon was chosen as the largest distance reached by 95% of the cells. The direction in which each cell first crossed the horizon is shown in the circular histogram (c). There is a significant preference for one direction (shown by the arrow) and the 95% confidence interval of the mean is shown by the shaded sector. The long axis of the aligned group of cells runs in this sector.
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