ISSN:0886-1544    

DOI:10.1002/cm.970240402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970240403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractHighly motile chick heart fibroblasts in primary culture (1° CHFs) gradually convert into much slower‐moving secondary (2°) 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°s and 2°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° CHFs and 60 min in 2° CHFs, with mainly MT fragments remaining beyond these times. All MTs and fragments were found to have turned over by 1 h in 1° CHFs and 80 min in 2°s. Because 2° CHFs were found to be on average six times larger than 1°s, the difference in MT turnover time was considered largely due to the size difference. For both 1° and 2° 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 α‐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° CHF cell shape, but rather play an ancillary role.

ISSN:0886-1544    

DOI:10.1002/cm.970240404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractTo 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 of125I‐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. © 1993

ISSN:0886-1544    

DOI:10.1002/cm.970240405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractNG108‐15 cells extend “rapid‐onset” neurites vigorously within the first hour after plating in minimal serum‐free medium on Petri dishes coated with polylysine and laminin (1 ng/mm2). We recently reported that the initial rates of neurite formation and cell translocation are further accelerated in this system when nonspecific substratum attachment sites are partially blocked by polyglutamate, bovine serum albumin, or polyethylene glycol polymers [Smalheiser, N. R. (1991): Dev. Brain Res. 62:81–89]. When cells were plated in the presence of the monovalent cation ionophore monensin (1–5 μM) or hypertonic sucrose (50–100 mM), the initial rate of outgrowth on laminin/polylysine‐treated Petri dishes was not affected, yet theaccelerationproduced by polyglutamate was strongly inhibited. These data indicate that monensin‐sensitive intracellular events can regulate neurite extension on laminin indirectly, through modulating the effects exerted on cells by nonspecific substratum sites. Although the critical events affected by monensin remain to be identified, movements of laminin receptors (their clustering, internalization, and recycling) are likely targets for further study. ©

ISSN:0886-1544    

DOI:10.1002/cm.970240406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractA procedure for preparing cytosol‐free ram sperm models was developed. Sperm are introduced to a Triton X‐100‐containing demembranation medium layered on top of a discontinuous Percoll gradient. After brief exposure to the demembranating solution, the sperm are separated from the detergent‐soluble components by centrifugation through a 55% Percoll layer, finally collecting on top of a 90% Percoll cushion from where they are recovered. Optimum conditions consisted of Triton X‐100 at 0.20% and a demembranation time of 35 sec. Cross‐sections of midpieces and principal pieces of the demembranated sperm were examined by electron microscopy. With 0.20% Triton X‐100 in the demembranation medium, 86% of the cross‐sections showed no plasma membranes and the rest had broken plasma membranes. The remaining tail structures appeared to be morphologically intact. Assay of phosphoglucose isomerase as a marker enzyme confirmed that at least 98% of the cytosolic protein was removed. Ram sperm models obtained by this procedure could be reactivated, and the percent motility and beat parameters were similar to those of the intact sperm. Reconstitution with the detergent‐soluble components was neither required for, nor enhanced, reactivation. Therefore, demembranated ram sperm do not require a detergent‐soluble protein factor for reactivation. ©

ISSN:0886-1544    

DOI:10.1002/cm.970240407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractA “freeze‐break” technique (Trombitás, K.: Acta Biochim. Biophys. Hung. 6:419–427, 1971) and immunoelectron microscopy were used to study the elastic properties of titin filaments. Small bundles of freshly prepared rabbit psoas muscle fibers were quickly frozen and broken under liquid nitrogen to fracture sarcomeres in planes perpendicular to the filament axis, in each of various regions along the sarcomere. The still‐frozen specimens were thawed during fixation to allow elastic filaments to retract. The broken specimens were then labelled with monoclonal anti‐titin antibodies against an unique epitope in the I‐band.The titin epitopes were normally positioned symmetrically about the Z‐line. However, in sarcomeres broken at the A‐I junction, the epitopes no longer remained symmetrical: the titin filaments in the broken half‐sarcomere retracted, independently of the thin filaments, forming a dense band just near the Z‐line. The retracted density apparently did not reach the Z‐line; retraction stopped at the level of the so‐called N1‐line. In sarcomeres broken at the Z‐line level, the titin filaments retracted in the opposite direction. In this case the titin epitope retracted all the way to the ends of the thick filaments.It appears then that titin molecules form elastic filaments that are independent of thin filaments in most of the I‐band. Near the Z‐line, however, the titin filaments either have an inelastic domain or associate firmly with the thin filaments at the N1

ISSN:0886-1544    

DOI:10.1002/cm.970240408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970240409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970240401

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY