Direct visualization of fluorescein-labeled microtubules in vitro and in microinjected fibroblasts.

139

111

Microtubule assembly in vivo was studied by hapten-mediated immunocytochemistry. Tubulin was derivatized with dichlorotriazinylaminofluorescein (DTAF) and microinjected into living, interphase mammalian cells. Sites of incorporation were determined at the level of individual microtubules by double-label immunofluorescence. The haptenized tubulin was localized by an anti-fluorescein antibody and a second antibody conjugated with fluorescein. Total microtubules were identified by anti-tubulin and a secondary antibody conjugated with rhodamine. Contrary to recent studies , J. Cell Biol., 99:2165-2174Saxton, W. M., et al., 1984, J. Cell Biol., 99:2175-2186 which suggest that tubulin incorporates all along the length of microtubules in vivo, we found that microtubule assembly in interphase cells was in vivo, as in vitro, an end-mediated process. Microtubules that radiated out toward the cell periphery incorporated the DTAF-tubulin solely at their distal, that is, their plus ends. We also found that a proportion of the microtubules connected to the centrosomes incorporated the DTAF-tubulin along their entire length, which suggests that the centrosome can nucleate the formation of new microtubules.

Section: Resultsmentioning

confidence: 96%

Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes.

Soltys¹,

Borisy²

1985

139

111

mentioning

confidence: 79%

“…In practice it is a useful method only if the labeled tubulin retains the functional characteristics of the native protein (17,18,36,40). Recent work supports the belief that the behavior of tubulin labeled with dichlorotriazinyl-aminofluorescein (DTAF) is analogous to the behavior of unlabeled tubulin (15,19,38,39).In this paper we report the use of DTAF-tubulin and recently developed techniques of fluorescent analogue cytochemistry (18, 30, 32, 35, reviewed in references 17, 34, 36, arid 4 l) to study in vivo tubulin dynamics by two different methods: the rate of incorporation of DTAF-tubulin by microtubules immediately following microinjection, and the rate of fluorescence redistribution after photobleaching (FRAP) of DTAF-tubulin after the incorporation process has proceeded to steady state. Both methods indicate that the apparent rate of exchange of free tubulin dimers with tubulin dimers in polymer is rapid in mitotic microtubules relative to interphase microtubules.…”

mentioning

confidence: 80%

“…The function of mitotic microtubules has been the subject of a great deal of debate for many years (reviewed in references 11 and 27), but it is clear that they are involved in the movement of chromosomes. Interphase cytoskeletal microtubules appear comparatively static with changes in arrangement and length occurring slowly (15). Although their function is also the subject of debate, they may be involved in the specification of cell polarity, shape, and cytoplasmic organization (3,22,28,37,44).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Tubulin dynamics in cultured mammalian cells.

Saxton¹,

Stemple²,

Leslie³

et al. 1984

469

323

Bovine neurotubulin has been labeled with dichlorotriazinyl-aminofluorescein (DTAF-tubulin) and microinjected into cultured mammalian cells strains PTK1 and BSC. The fibrous, fluorescence patterns that developed in the microinjected cells were almost indistinguishable from the pattern of microtubules seen in the same cells by indirect immunofluorescence. DTAF-tubulin participated in the formation of all visible, microtubule-related structures at all cell cycle stages for at least 48 h after injection. Treatments of injected cells with Nocodazole or Taxol showed that DTAF-tubulin closely mimicked the behavior of endogenous tubulin. The rate at which microtubules incorporated DTAF-tubulin depended on the cellcycle stage of the injected cell. Mitotic microtubules became fluorescent within seconds while interphase microtubules required minutes. Studies using fluorescence redistribution after photobleaching confirmed this apparent difference in tubulin dynamics between mitotic and interphase cells. The temporal patterns of redistribution included a rapid phase (~3 s) that we attribute to diffusion of free DTAF-tubulin and a second, slower phase that seems to represent the exchange of bleached DTAF-tubulin in microtubules with free, unbleached DTAF-tubulin. Mean half times of redistribution were 18-fold shorter in mitotic cells than they were in interphase cells.The organization and apparent function of microtubules in cultured mammalian cells vary markedly as the cell cycle proceeds from interphase to mitosis and back to interphase again. Structural studies with polarization optics, immunofluorescence, and electron microscopy have shown that the mitotic spindle is an active structure in which microtubules exhibit rather rapid changes in arrangement and length (l 1-13, 22, 23, 27, 3 l, 38). The function of mitotic microtubules has been the subject of a great deal of debate for many years (reviewed in references 11 and 27), but it is clear that they are involved in the movement of chromosomes. Interphase cytoskeletal microtubules appear comparatively static with changes in arrangement and length occurring slowly (15). Although their function is also the subject of debate, they may be involved in the specification of cell polarity, shape, and cytoplasmic organization (3,22,28,37,44).To understand the different behaviors of microtubules in mitotic and interphase cells, it may be useful to investigate the dynamic relationship between tubulin and microtubules in vivo. Studying this relationship in detail requires a method for viewing tubulin in both polymer and dimer form in living cells. In principle, microinjection of fluorescently labeled tubulin followed by fluorescence microscopy provides such a method. In practice it is a useful method only if the labeled tubulin retains the functional characteristics of the native protein (17,18,36,40). Recent work supports the belief that the behavior of tubulin labeled with dichlorotriazinyl-aminofluorescein (DTAF) is analogous to the behavior of unlabeled tubulin (15,19,...

“…5). These specific tests for the fidelity of injected H1, similar demonstrations'of expected behavior for HMG proteins in previous microinjection studies (43), and the observation that various derivatized cytoskeletal proteins can assemble into actin filaments or microtubules (9,18) indicate that proteins labeled at a few residues can retain a high degree of native structure. Consequently, we believe that injected 3H-histone H1 is a valid probe for the behavior of its endogenous counterpart.…”

Section: Discussionmentioning

confidence: 92%

Dynamic behavior of histone H1 microinjected into HeLa cells.

Wu¹,

Kuehl²,

Rechsteiner³

1986

Abstract. Histone HI was purified from bovine thymus and radiolabeled with tritium by reductive methylation or with 1251 using chloramine-T. Red blood cell-mediated microinjection was then used to introduce the labeled H1 molecules into HeLa cells synchronized in S phase. The injected HI molecules rapidly entered HeLa nuclei, and a number of tests indicate that their association with chromatin was equivalent to that of endogenous histone H1. The injected molecules copurified with HeLa cell nucleosomes, exhibited a half-life of ~o100 h, and were hyperphosphorylated at mitosis. When injected HeLa cells were fused with mouse 3T3 fibroblasts <10% of the labeled H1 molecules migrated to mouse nuclei during the next 48 h. Thus, the intracellular behavior of histone H1 differs markedly from that of high mobility group proteins 1 and 2 (HMG1 and HMG2), which rapidly equilibrate between human and mouse nuclei after heterokaryon formation (Rechsteiner, M., and L. Kuehl, 1979, Cell, 16:901-908; Wu, L., M. Rechsteiner, and L. Kuehl, 1981, J. Cell Biol, 91: 488-496). Despite their slow rate of migration between nuclei, the injected H1 molecules were evenly distributed on mouse and human genomes soon after mitosis of HeLa-3T3 heterokaryons. These results suggest that although most histone H1 molecules are stably associated with interphase chromatin, they undergo extensive redistribution after mitosis. I x is generally accepted that histones are arranged as nucleosomal arrays consisting of two molecules each of histones H2A, H2B, H3, and H4, around which are wrapped ,o140 base pairs (bp) of DNA. There is considerable support for this model: SDS polyacrylamide gel analysis shows the four core histones to be present in roughly equal amounts; gel filtration and cross-linking studies indicate specific histone interactions; limited nuclease digestion generates discrete particles containing the four core histones and 145 bp of DNA, electron microscopic observations show repeating knoblike structures along histone HI-depleted chromatin fibers; and x-ray diffraction patterns confirm the basic octamer structure (13,19,24,29; see reference 15 for review).There is also wide agreement that histone HI serves to coil the nucleosomal thread into higher order, more compact structures (28, 34). The exact arrangement of nucleosomes in the thicker fibers produced by histone HI remains an area of active study, and evidence has been presented for solenoid (7,23,40), superbead (32, 44), or zig-zag ribbon arrangements (41, 42; see reference 6 for review). Several recent observations indicate that histone HI binding to chromatin can have important consequences for transcription. Xenopus 5S genes are protected from histone Hi-mediated repression by the presence of active transcription complexes, and prior H1 binding prevents formation of such stable transcription complexes (31). Similarly, fractionation of avian red blood cell (RBC) ~ chromatin produces discrete supranucleosomal particles enriched in inactive gene sequences, and it has been suggest...