Cell motility and the cytoskeleton最新文献

Extraction of Dictyostelium amoebae with Triton X-100 produces robust cytoskeletons composed mainly of actin and myosin II. These cytoskeletons rapidly contract when mixed with Mg-ATP in simple buffers. The Triton-soluble fraction was found to contain a GTP-dependent activity that prevented contraction by Mg-ATP. This activity was purified, and identified, as nucleoside diphosphate kinase (NDP kinase). The apparent inhibition resulted from pre-contraction of the cytoskeletons. Tightly bound cytoskeletal ADP was presumably phosphorylated, and the resulting ATP powered contraction. NDP kinase appeared to be unique in this capacity, since other regenerating systems did not cause pre-contraction. Reconstitution experiments demonstrated that the kinase must be in physical contact with the cytoskeleton. These results suggest that Dictyostelium NDP kinase is able to channel ATP to the myosin molecule, and this could play a role in directly regulating cytoskeletal contraction or in facilitating contraction under conditions where intracellular ATP concentrations are low. This ability to modulate cytoskeletal contraction could help to explain observations in other systems whereby defects in NDP kinase result in abnormal development or changes in the metastatic potential of cancer cells.

The molecular cloning and sequencing of microtubule-associated protein (MAP)-4 identified microtubule-binding repeats near the C-terminus and a projection domain near the N-terminus. Although it is well known that MAP-4 stimulates the assembly of and stabilizes microtubules (MT) in vitro, the function of MAP-4 in vivo is still unclear. In this study, we examined the function of MAP-4 in the cytoskeleton both in vitro and in vivo. Intact MAP-4 was prepared from bovine adrenal cortex, and the truncated fragments of the N- and the C-terminal halves (named NR and PA4 fragments, respectively) were expressed in Escherichia coli and isolated. In vitro studies demonstrated that in a solution containing a physiological concentration of NaCl, intact MAP-4 and the PA4 fragment were bound to MT, but not to F-actin. The NR fragment was not bound to MT or to F-actin. We also examined the MT changes in PtK2 cells after they had been microinjected with intact MAP-4 and the truncated fragments of PA4 and NR. The injection of intact MAP-4 or PA4 into the cells induced an increase in the number of cytoplasmic MT, as well as MT bundling. The NR fragment did not affect the MT array. Injected MAP-4 and PA4 were associated with the increased MT. In addition, injection with MAP-4 and PA4 stabilized MT in relation to treatment with the MT-disrupting drug, nocodazole. These results indicated that intact MAP-4 and the PA4 fragment promoted MT assembly and stabilized MT, by binding to MT, in vivo as well as in vitro. Further, the injection of the PA4 fragment induced an increase in stress fibers. However, these proteins did not show any association with the stress fibers. Our results suggest that there is an indirect effect of MAP-4 on stress fibers rather than a direct interaction between MAP-4 and stress fibers.

A panel of monoclonal antibodies (mAbs) has been generated against sea urchin sperm axonemes and selected for their ability to inhibit the motility of sea urchin sperm models. The mAb C9 recognized a 50 kDa protein on blots of sea urchin sperm axonemes. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that C9 recognized isoforms of beta-tubulin. Low concentrations of C9 (0.1-1.0 microgram/ml) blocked the motility of sea urchin sperm models by decreasing the sliding velocity and frequency of flagellar beating to less than 1 Hz and by modifying the shear angle along the axoneme, especially the distal end. Other antitubulin antibodies had little effect on motility at concentrations 100-fold higher than those effective for C9. The effects on motility were not restricted to flagella of sea urchin spermatozoa. Flagellar beating of the dinoflagellate Oxyrrhis marina was completely blocked by C9 in a manner reminiscent of that of sea urchin sperm flagella. The mAb also inhibited the motility of human spermatozoa and Chlamydomonas reinhardtii. Immunofluorescence techniques revealed that C9 stains the whole axoneme of sea urchin spermatozoa and O. marina flagella together with the cortical network of O. marina cell body. C9 is the first antitubulin antibody reported to interfere with flagellar beat frequency. The observation that this antibody arrests the motility of flagella from sea urchin sperm along with that of dinoflagellate, algae, and human sperm flagella suggests that the epitope recognized by C9 is conserved over a long period of evolution and plays an important role in sperm motility.

Studies on the flagellar movement of carp spermatozoa induced by dilution in distilled water allowed us to describe a sequence of early, rapid morphological and kinetic changes which begin at the very tip of the flagellum. They cause the progressive folding of the axoneme which ends stuck to the head within 90-120 seconds after the initiation of motility. However, the axonemal machinery remains functional as the folding can be reversed after transfer back into a high osmolality medium and partially folded flagella were able to propagate efficient waves along the non-folded proximal portion of the axoneme. The data also revealed that the membrane area of the terminal piece exhibits strong sensitivity to hypotonicity. These results suggest that in the normal freshwater medium, the brief swimming period allowing fertilization of oocytes is limited by the osmotic stress induced coiling of the carp sperm tail and not by ATP stores.

It has been demonstrated that the activity of ADF and cofilin, which constitute a functionally related protein family, is markedly altered by phosphorylation, and that the phosphorylation site is Ser 3 in their amino acid sequences [Agnew et al., 1995: J. Biol. Chem. 270:17582-17587; Moriyama et al., 1996: Genes Cells 1:73-86]. In order to clarify the function of the phosphorylated and unphosphorylated forms of cofilin in living cells especially in the process of cytokinesis, we generated analogs of the unphosphorylated form (A3-cofilin) and phosphorylated form (D3-cofilin) by converting the phosphorylation site (Ser 3) of cofilin to Ala and Asp, respectively. The mutated proteins were produced in an Escherichia coli expression system, and conjugated with fluorescent dyes. In in vitro functional assay, labeled A3-cofilin retained the authentic ability to bind to and sever F-actin, while labeled D3-cofilin failed to interact with actin. They were then injected into living cells to examine their cellular distribution. They exhibited distinct localization patterns in the cytoplasm; A3-cofilin was highly concentrated at the membrane ruffles and cleavage furrow, where endogenous cofilin is also known to be enriched. In contrast, D3-cofilin showed only diffuse distribution both in the cytoplasm and nucleus. These results suggest that the subcellular distribution of cofilin as well as its interacting with actin in vivo is regulated by its phosphorylation and dephosphorylation.

Cellular contractility plays an important role in cell morphogenesis and tissue pattern formation. In this context, we examined how the expression of cell traction depends on cell-to-substrate contacts and cytoskeletal organization. Qualitative observation of chick fibroblasts cultured on an elastic film of polydimethylsiloxane indicated a strong spatial relationship between wrinkle pattern and distribution of actin stress fibers and focal contacts. In order to further characterize cell contractility, the projected area of Triton-permeabilized fibroblasts upon ATP-induced retraction was measured in various conditions of substrate adhesivity, cytoskeletal perturbation, and temperature. In all conditions, the relationship between degree of cell retraction and ATP concentration was well described by the laws of enzyme kinetics. Culturing cells on a gelatin-coated substrate, decreasing the temperature, using phosphate ribonucleotides other than ATP, and treating cells with cytochalasin D all diminished the rate of cell retraction, indicating that fibroblast traction is generated by a temperature- and ATP-dependent actin/myosin stress fiber sliding mechanism, transmitted to the substrate through focal adhesions. Treatment of cells with either nocodazole or taxol did not affect retraction of permeabilized fibroblasts upon stimulation with ATP, suggesting that microtubules do not directly resist cell traction. Treatment of cells with vanadate increased cell retraction, suggesting that intermediate filaments help transmit tension.

To explore the behaviour of microtubule-associated proteins, MAP2 and TAU in the interactions of mitochondria with microtubules, an homologous acellular system has been reconstituted with organelles isolated from rat brain. We have established a quantitative in vitro binding assay based on the cosedimentation of 125I-labeled microtubules with mitochondria. We found that binding of microtubules to mitochondria was concentration dependent and saturable. Binding was insensitive to ATP. A comparison of taxol-stabilized microtubules prepared from MAP-free tubulin or tubulin coated with TAU or MAP2 showed that the microtubule-associated proteins diminished, or reduced to background levels, the formation of complexes with mitochondria. In contrast, the amount of MAP-free taxol microtubules that cosedimented with mitochondria increased two- and six-fold when mitochondria were coated with MAP2 or TAU. These studies suggest that the two major brain MAPs could have a crosslinking or a spacing role, depending on their organelle localization.

Highly motile chick heart fibroblasts in primary culture (1 degree CHFs) gradually convert into much slower-moving secondary (2 degrees) cells. The polarized movement of the latter, but not the former, cell type has been found to be dependent on an intact microtubule (MT) network [Middleton et al., 1989, J. Cell Sci. 94:25-32]. To investigate the comparative stability of the MT networks of 1 degree s and 2 degrees s, turnover was investigated by microinjection of biotin-labeled brain tubulin to act as a reporter. MTs in both cell types were found to be very dynamic, with the MT networks effectively disassembled by about 30 min in 1 degree CHFs and 60 min in 2 degrees CHFs, with mainly MT fragments remaining beyond these times. All MTs and fragments were found to have turned over by 1 h in 1 degree CHFs and 80 min in 2 degrees s. Because 2 degrees CHFs were found to be on average six times larger than 1 degree s, the difference in MT turnover time was considered largely due to the size difference. For both 1 degree and 2 degrees cells, the more slowly turning over MTs were generally curly and perinuclear in distribution, resembling stable MTs in other systems, but they appeared significantly earlier in CHFs. However, no discrete subpopulations of slower turning over MTs were found to be associated with either the leading edges or the processes of either cell type. In addition, no major differences were identified in the patterns of modified alpha-tubulin along the MTs or of MT cold or drug stability. It is concluded that MTs do not have a direct structural or skeletal function in maintaining a polarized 2 degrees CHF cell shape, but rather play an ancillary role.