Peter Sheterline

Localisation of the major high-molecular-weight protein on microtubules in vitro and in cultured cells

Abstract An antiserum has been produced which is specific for the major high-molecular-weight protein (HMW) associated with pig brain microtubules assembled in vitro. The HMW protein can be localised on the surface of brain microtubules assembled in vitro as a corase helix by using a peroxidase labelling technique. In cultured ovarian granulosa cells, by using indirect immunofluorescence, the HMW is present on cold- and colchicine-sensitive structures which have an intracellular distribution similar to microtubules. It seems likely, therefore, that the brain protein is representative of a class of proteins associated with non-neuronal cytoplasmic microtubules.

Trifluoperazine can distinguish between myosin light chain kinase-linked and troponin C-linked control of actomyosin interaction by Ca++

Summary Trifluoperazine specifically inhibits both the increased ATPase and the degree of phosphorylation of myosin light chains induced by 5 × 10−5M Ca++ in chicken gizzard muscle actomyosin preparations. Half maximal inhibition occurs at a concentration of trifluoperazine of 3 × 10−6M. Pig skeletal muscle actomyosin ATPase was not inhibited by concentrations of trifluoperazine below 10−4M. It is suggested that trifluoperazine can be used to distinguish between the two different mechanisms of Ca++ regulation of actomyosin interaction, operational in smooth and skeletal muscle, and can thus be used to investigate the class of Ca++ control of actomyosin operating in various aspects of non-muscle cell motility.

Phorbol ester induces rapid actin assembly in neutrophil leucocytes independently of changes in [Ca2+]i and PHi

SummaryThe phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), at nanomolar concentrations, induces rapid (t1/2 approximately 30 s) protrusion of multiple petal-shaped lamellae by neutrophil leucocytes. Lamellae are richly endowed with actin filaments as determined by the localization of rhodamine-phalloidin, suggesting extensive assembly at the cell cortex. Direct measurement of the proportion of total cell actin which is polymerized, by using a deoxyribonuclease I inhibition assay, indicates that the proportion of polymerized actin approximately doubles, and that assembly initiated by 30nm TPA occurs with no obvious lag phase and with a t1/2 of about 30 s. A half-maximal response was induced at 2nm TPA. Since both actin assembly and protrusion of lamellae are completely inhibited by 10−6m cytochalasin D, protrusion of lamellae is presumably dependent on actin filament assembly.To examine whether TPA induces actin assembly via changes in [Ca2+]i or pHi, these parameters were monitored in cells loaded with the fluorescent indicators quin2 and quene1 respectively. Addition of TPA caused no change in [Ca2+]i but a biphasic change in pHi. To examine further the potential role of ionic changes in regulation of actin assembly, the morphological responses of cells to TPA were monitored in severely Ca2+-depleted cells, or cells in which pHi had been experimentally raised or lowered by simultaneous additions of a weak base (NH4Cl) or weak acid (CH3COONa) respectively. The protrusion of lamellae induced by TPA was completely unaffected by these experimental manipulations indicating that TPA can directly regulate actin assembly, and hence morphology, by mechanisms essentially independent of [Ca2+]i and pHi. Since TPA is believed to mimic diacylglycerol, which is putatively generated by cleavage of phosphatidylinositol-4, 5-bisphosphate in a number of cellular responses involving actin filament assembly, these results implicate diacylglycerol, either acting directly, or via protein kinase C-mediated phosphorylation, as a common step in the receptor-mediated regulation of cortical actin filament assembly in cells.

Cell shape-dependent Control of Ca2+ influx and cell cycle progression in Swiss 3T3 fibroblasts.

The ability of adherent cells such as fibroblasts to enter the cell cycle and progress to S phase is strictly dependent on the extent to which individual cells can attach to and spread on a substratum. Here we have used microengineered adhesive islands of 22 and 45 μm diameter surrounded by a nonadhesive substratum of polyhydroxyl methacrylate to accurately control the extent to which individual Swiss 3T3 fibroblasts may spread. The effect of cell shape on mitogen-evoked Ca2+ signaling events that accompany entry into the cell cycle was investigated. In unrestricted cells, the mitogens bombesin and fetal calf serum evoked a typical biphasic change in the cytoplasmic free Ca2+ concentration. However, when the spreading of individual cells was restricted, such that progression to S phase was substantially reduced, both bombesin and fetal calf serum caused a rapid transient rise in the cytoplasmic free Ca2+ concentration but failed to elicit the normal sustained influx of Ca2+ that follows Ca2+ release. As expected, restricting cell spreading led to the loss of actin stress fibers and the formation of a ring of cortical actin. Restricting cell shape did not appear to influence mitogen-receptor interactions, nor did it influence the presence of focal adhesions. Because Ca2+ signaling is an essential component of mitogen responses, these findings implicate Ca2+ influx as a necessary component of cell shape-dependent control of the cell cycle.

Cell cycle‐dependent morphological changes in the actin cytoskeleton induced by agents which elevate cyclic AMP

Agents which increase the intracellular concentration of cyclic adenosine‐5′‐monophosphate, induce a highly arborised morphology in a proportion of sub‐confluent Swiss 3T3 fibroblasts. During this process the organisation of actin filaments progessively changes from a characteristic stress fibre pattern to leave a network of actin filaments within each and every arborisation. Despite this massive reorganisation of the actin cytoskeleton no changes are observed in the extent of polymerisation of actin during arborisation. The proportion of cells in asynchronous cultures undergoing arborisation at maximal concentrations of agents reaches a maximum of 30%; suggesting that the effect might be mediated only in cells during a restricted period of the cell cycle. More than 80% of serum‐starved cells responded to these agents between 1 and 8 hours after readdition of serum, but not at other times, suggesting that the arborisation response can occur only in the G1 phase of the cell cycle.

Immunological characterization of the microtubule-associated protein map2

When microtubules are assembled from mammalian brain extracts in vitro, several proteins other than tubulin copurify in constant stoicheiometry to tubulin as judged by SDS-electrophoresis [l]. Among these microtubule-associated proteins are two or more high molecular weight polypeptides with chain weights >2.50 000 and a group of polypeptides (tau) with chain weights of 55 000-70 000. Both a 280 000 mol. wt polypeptide(s), designated MAP, and the tau proteins apparently share the ability to increase the rate of assembly of pure tubulin in vitro [2]. and further, both proteins have been shown to be present along the length of microtubules in situ by using specific antibodies [3,4]. It thus seems likely that some of these proteins at least are integral parts of the functional microtubule. MAP, is a particularly interesting protein since it appears to be the major component of the side arms of microtubules seen by both negative staining and by transmission electron microscopy [2] where it appears to form an organised lattice on the microtubule surface [5.6]. Most models relating to the function of microtubules in cells imply interaction between microtubules and other subcellular organelles (see [S] for discussion), thus the role of a side arm protein may be crucial for the specificity and modulation of such an interaction. Indeed there is some evidence that microtubule-associated proteins are necessary for the interaction in vitro between actin filaments [7] or secretory granules [8] and microtubules. It may also be pertinent that MAP? is the preferred substrate for a microtubule-associated cyclic AMPdependent protein kinase [9], although there is no