Microscopy microtubules

Figure 3.7 Application of the probe for demonstrate the increased resolution of CHI-super-resolution microscopy microtubules RON, which becomes even clearer when in fixed cells were labeled with 2a and looking at the intensity profiles (c) along...

When tubulin heterodimers are assembled into microtubules, they form linear protofilaments with the P-tubulin subunit of one tubulin molecule linking covalently with the a-subunit of the next. Direct examination by electron microscopy of tannic acid-treated specimens has shown that micrombules in neurons and the A-microtubules of cilia and flagella have 13 protofilaments arranged side to side to form a cylinder around what appears to be an empty lumen. 

Figure 7. Video-enhanced DIC microscopy of rat liver Golgi apparatus membrane networks moving along microtubules using Xenopus egg microtubule motors (Allan and Vale, 1994). Top panel membrane extension with a bulbous terminus (arrow) attached to a microtubule (arrow heads). Middle panel same field two seconds later. The membrane has advanced about 3 pm along the microtubule (arrow). Bottom panel membrane has now advanced further along the microtubule (arrow). Bar = 2 pm.

The processes of meiosis and mitosis involve many motile events, from the separation of the daughter chromosomes to the final act of cell separation at cytokinesis (Wadsworth, 1993). DNA replication itself may be considered as a motile event, because the polymerase complex moves along the linear DNA. One of the most obvious motile events is the separation of the chromosomes along the mitotic spindle at anaphase. Details of the structure and polarity of microtubules in the spindle apparatus in meiosis and mitosis are known through electron and light microscopy, but it is not yet clear whether the chromosomes are pushed, pulled or... 

Until the past decade, the cytoplasm was widely considered to be structurally unorganized with the main division of labor at the organellar level. Certainly, relatively little was known about the nature of the cyto-skeleton (with the notable exception of the mitotic apparatus and striated muscle), and the dynamics of cytoplasmic behavior were conceptualized vaguely in terms of sol-gel transitions without a sound molecular foundation. Substantial improvements in electron, light, and fluorescence microscopy, as well as the isolation of discrete protein components of the cytoskeleton, have led the way to a much better appreciation of the structural organization of the cytoplasm. Indeed, the lacelike network of thin filaments, intermediate filaments, and microtubules in nonmuscle cells is as familiar today as the organelles identified... 

Wick, S. M., Seagull, R. W., Osborn, M., Weber, K., and Gunning, B. E. S. (1981) Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells. J. Cell Biol. 89, 685-690. 

The IC, values of the vinca binary alkaloids for microtubule assembly were measured from their concentration-dependent effects on steady-state turbidity levels. Values are presented from two separate experiments. The products induced by a high concentration (10 M) of each compound with MTP or steady-state microtubules assembled from MTP were determined by transmission electron microscopy of steady-state solutions from at least two separate preparations of protein with similar results. SA, Spiral aggregates S, single spirals Am. amorphous aggregates MT, microtubules N.D., not determined. 

Fig. 4. Transmission electron microscopy of MTP reaction mixtures, (a) An opened area is seen in a microtubule polymerized in the presence of an IC concentration (3 x 10 W) of deoxydesethyl VBL (5). (b) Single spirals are formed from MTP and 10 M 5. (c) Spiral aggregates are formed from MTP and 10 M VBL. (d) MTP incubated with an lCs concentration (2 X 10 Af) of epimethyldeoxydesethyl VBL (4) formed spiral aggregates both free in solution (single arrows) and associated with microtubules (double arrows), (e) Greater magnification of MTP incubated with an 1C , concentration (2 x 10 M) of methyldeoxyde-sethyl VBL (3) displays a free spiral (arrow) and spiralized material on the microtubules. Bar, 0.1 p.m.

Video microscopy has permitted direct observation of microtubule assembly/disassembly dynamics in vitro. Horio and HotanP first used dark-field optics to observe the growth and shrinkage phases, but so-called Allen video-enhanced contrast microscopy has become most convenient. 

Dombeck, D. A., Kasischke, K. A., Vishwasrao, H. D., et al. 2003. Uniform polarity microtubule assemblies imaged in native brain tissue by second harmonic generation microscopy. Proc. Natl. Acad. Sci. USA 100 7081. 

Electron microscopy reveals several types of protein filaments crisscrossing the eukaryotic cell, forming an interlocking three-dimensional meshwork, the cytoskeleton. There are three general types of cytoplasmic filaments— actin filaments, microtubules, and intermediate filaments (Fig. 1-9)—differing in width (from about 6 to 22 nm), composition, and specific function. All types provide structure and organization to the cytoplasm and shape to the cell. Actin filaments and microtubules also help to produce the motion of organelles or of the whole cell. 

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