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doi: 10.1242/10.1242/jcs.00062

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Osteoclast spreading kinetics are correlated with an oscillatory activation of a calcium-dependent potassium current

INSERM Unit 403, Hôpital E. Herriot, Pavillon F, 69437 Lyon Cedex 03, France

View larger version (53K): [in a new window]   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.

View larger version (53K): [in a new window]   Fig. 8. Correlation analysis between membrane spreading kinetics and whole-cell current. (a) A polar diagram of the cell perimeter during spreading; the red and blue polygons correspond to the initial and final states of the cell, respectively. (b) A polar representation of the correlation coefficient between membrane current and each axial rate of membrane spreading. The correlation was higher for axes where the movements' amplitude was larger and lower for zones with slight moves or retractile displacements. (c) The red line represents the global spreading rate calculated with multiple regression analysis and the black line the membrane current. This shows a very significant correlation between multiple regression of spreading rates and IKCa current. (d) Coherence analysis between membrane current and membrane spreading rate; the peaks indicate the presence of frequencies for which both phenomena are in phase.

View larger version (27K): [in a new window]   Fig. 2. A spontaneous oscillating current is activated during osteoclast spreading. Osteoclast background current was continuously monitored by voltage ramps applied every 2 seconds. The spontaneous outward oscillating current (measured at 0 mV) exhibited two patterns, depending on the cells, a periodic rhythmic activation (a) or variable inter-peaks intervals (b). The periodicity of the current signal was tested by the auto-correlation function. (c) Periodic function resulting from a simple periodic signal. (d) Complex pattern resulting from a non-periodic complex signal. (e) 19 out of 20 spreading cells showed a positive oscillating current during spreading, whereas 16 out of 20 non-spreading cells did not exhibit any current.

View larger version (17K): [in a new window]   Fig. 3. The ionic current activated during osteoclast spreading is a potassium current. (a) Currents were recorded with a voltage ramp protocol (insert). (a) Current-voltage (I-V) relationships measured at the peak of oscillation and at the baseline. (b) Current voltage relationship of the difference current (peak—baseline) (Erev=-75 mV, [K+]o=5 mM). (c) I-V curves of the difference current measured in the presence of different external potassium concentrations. Erev shifted to positive potentials when external potassium increased. (d) Plot of Erev versus Log of the external potassium concentration. The linear regression gave a slope of 57.8 mV per decade, in agreement with a potassium current.

View larger version (20K): [in a new window]   Fig. 4. Effect of an internal Ca2+ increase on the osteoclast background current. (a) Effect of 10 µM calcium ionophore A-23187 on holding current (Vholding=-20 mV). The internal calcium rise induced the activation of an outward current `oscillation'. (b) I-V relationships of the current before and after application of Ca2+ ionophore. (c) I-V curve of the difference current (A-23187—control). Characteristics of this current were identical to those of the current induced during spontaneous oscillations. (d) Inside-out patch recording showing channel activation by 0.5 mM Ca2+ applied at the inner face of the patch membrane. Activation of this channel induced burst openings made by fast switching between close and open states (see magnification in panel d). (Mean unitary conductance was =25±2 pS.)

View larger version (21K): [in a new window]   Fig. 5. Activation of a single channel during osteoclast spreading. Spontaneous electrophysiological activity was studied at single channel level during osteoclast membrane spreading. (a) For cell-attached recording, the pipette tip was located in the cell nuclei area (nuclei are represented by dotted circles). (b) Burst openings of a single channel with a unitary conductance of 22 pS (transmembrane potential=-80 mV). (c) Time evolution of the open probability for the observed channel. The open probability peaks (dotted box) correspond to the channel burst openings.

View larger version (19K): [in a new window]   Fig. 6. Pharmacology and role of the KCa current on bone resorption. (a) 10 µM of the calcium ionophore A-23187 induced a reversible increase of the outward current (IKCa) that was completely and reversibly inhibited by 10 nM charybdotoxin (CTX) and partially blocked in an irreversible manner by 0.5 nM Apamin (Apa). (b) CTX and APA incubated during 24 hours with osteoclasts cultured on glass coverslips induced a significantly lower mean osteoclasts area (n70). (c) Bone resorption in vitro: CTX and APA were incubated during 24 hours with osteoclasts cultured on cortical bone slices. Both specific blockers of KCa channels inhibited the number of resorption pits compared with the control.

View larger version (125K): [in a new window]   Fig. 7. Spread osteoclasts exhibit different spreading patterns. Membrane spreading is illustrated for three osteoclasts. The right image shows the cell appearance at the beginning of time-lapse recording, and the left image illustrates the cell at the maximum spreading. During this process, some osteoclasts showed a `symmetrical' spreading by radial axes around the nuclei (N) (cell 1), whereas other cells only exhibited membrane movement along a few directions (cells 2 and 3). Membrane spreading may take place over 180° opposite directions, as is seen in cell 3.