Microtubule dynamics in living cells: direct analysis in the internal cytoplasm.

Abstract

Microtubules (MTs) are long non-covalent polymers of heterodimeric tubulin that exchange with the soluble pool exclusively from their ends. The two opposite ends of MT are different: one end of the MT (plus end) is more dynamic than the opposite (minus) end. At steady state, plus ends of individual MTs undergo long excursions of growth and shortening. In many animal cells, MTs run from the cell center towards the periphery and form a radial array. Minus ends of MTs are located in the center while plus ends are directed presumably towards the cell margin and display dynamic instability behavior. In the interphase cell, the length of MTs in the radial array is seemingly limited by the cell margin, yet their plus ends are dynamic. How does the cell keep an array of long and dynamic MTs? To evaluate the formation and turnover of long MTs, it is necessary to follow individual MT not only at the cell periphery, but also deep in the cytoplasm. Direct observation of MTs in the internal cytoplasm is difficult because of the limited resolution of the light microscope. Recently, we developed experimental approaches that allow observation of MT dynamics in the areas where density of MTs is high (Komarova et al., 2002; Vorobjev et al., 1999). Using these approaches, MT dynamics in CHO and NRK cells were found to be different in the internal cytoplasm from those near the cell margin. Nascent MTs grew persistently from the centrosome towards the cell margin and displayed dynamic instability only near the margin (Komarova et al., 2002). Is this behavior common among animal cells? Addressing this question in the present study, we evaluated MT dynamics in the internal cytoplasm of several cultured celltypes (fish keratocytes, PtK1 cells; NIH-3T3 cells and REF fibroblasts) using the following experimental approaches: image processing that enhances contrast of individual MTs; sequential subtraction analysis to follow MTs elongation and growth; and laser bleaching of fluorescence. Cells cultured on the coverslips were microinjected with Cy-3 labeled tubulin and time lapse sequences were obtained on the inverted microscope using a cooled CCD camera as described elsewhere (Vorobjev et al., 1997). In keratocytes, MT density is very low and growth from the centrosome was analyzed directly after image processing. Nascent MTs grew from the centrosome persistently up to two-third of the cell radius (15–20 μm) and started to oscillate only after reaching cell periphery (outer one-third of cell radius). In PtK and REF cells, growth from the centrosome was analyzed during recovery after photobleaching. A nearly rectangular bleached zone, 5–8 μm wide and 40–50 μm long, was created using an argon laser focused through a cylindrical lens and a 100 objective lens on a Nikon Eclipse inverted microscope as described elsewhere (Keating et al., 1997). To obtain the wide bleached zone, the laser beam was directed onto the objective lens off-center of the main optical axis of the microscope. In the bleached zone, individual MTs could be traced for 1–3 min until the density of fluorescent MTs recovered to the high level, precluding further observation. MTs in the internal cytoplasm grow and shorten without pause in keratocytes, 3T3 and REF cells. Meanwhile in PtK cells, pauses in the internal cytoplasm were observed frequently. Shortening of MTs in all types of cells was non-processive. In fish keratocyte, length of shortening was 5.7 4.4 μm (one-third of cell radius), and could be approximated by gamma-distribution. Growth periods of MTs from the centrosome were rather long, and about 10% of MTs in 3T3, REF and * Corresponding author. Tel./fax: +7-(095)-939-2084. E-mail address: ivorobjev@mailru.com (I.A. Vorobjev). Cell Biology International 27 (2003) 293–294 Cell Biology International