Fig. 1. Osteoclast membrane movement analysis. (a) Osteoclast (OC) cultured on a glass coverslip. To obtain membrane movement analysis, the cell geometric centroide was calculated and taken as the origin point for a 30° angular division of the field. This cell showed forming lamellipodia in regions from 90° to 270°. Cell spreading was analysed along the 12 axes. (b) This image illustrates the evolution of membrane edge at 180°, which corresponds to the border (arrows) between the homogeneous grey region of the coverslip and refringency variations within the cell (see Materials and Methods). (c) Time evolution curve of the membrane edge derived from panel b. (d) The spreading rate time course was extracted from graph c by differentiation. The movement rate showed peaks of 0.75 µm/second for a mean spreading rate in that direction of 0.1 µm/second. (e) Movement map of the membrane edge for the 12 axes. The spreading rate along each axis was used to build up a matrix. Isocontours of the normalised spreading rate were drawn to visualise zones of identical values. Spreading occurred by isolated peaks (green to red) surrounded by periods of rest (light blue) or retraction (blue to deep blue). The movement map summarises, into `one image', the cell edge movement during the whole time lapse recording, along the cell perimeter. Two continuous `waves' of spreading were observed for this cell: one started between 270° to 240° at time 450 seconds, turning clockwise until 60° to 90° at time 1000 seconds, followed by a retraction (240° at time 700 s to 90° at time 1100 seconds). A second started at 240°-270°, at time 850 seconds, to 150° at time 1250 seconds. The bottom trace shows the background current for this cell recorded during spreading.

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