Journal of cell science. Supplement最新文献

Short microtubule seeds are constructed using heavily rhodamine-labeled tubulin. Polymerisation off the ends of these seeds is initiated using a mixture of 1:10 labeled and unlabeled tubulin, so that the new polymerisation is only dimly labeled. This is done in the presence of NEM tubulin, which inhibits growth from the microtubule minus ends. The polarity-marked microtubules are fixed at a desired length by adding taxol.

Cell motility events require movement of the cytoskeleton. Actin-based movement is catalyzed by the mechanoenzyme myosin, which translocates toward the barbed end of actin filaments in an ATP-dependent fashion. There are two subclasses of myosin with different structures and functions: conventional filamentous myosin (myosin II) and monomeric myosin I. Vertebrate non-muscle myosins I and II function as similar actin motors in vitro, catalyzing virtually identical actin-activated MgATP hydrolysis and motility. The functional diversification of these two enzymes results from their differential regulation. Calcium and tropomyosin, which activate the MgATP hydrolysis and motility of vertebrate non-skeletal muscle myosin II proteins, inhibit vertebrate (brush border) myosin I. The activities and regulation of brush border myosin I provide insight into conserved and unique features of the myosin mechanoenzymes and suggest how the functions of myosins I and II are divided in vertebrate cells. Brush border myosin I as an enzyme also contributes to our understanding of the molecular mechanism of motility.

Studies of the molecular mechanism of motile activity require the capacity to examine the properties of individual, isolated molecular components and the properties of these same molecular components in the organised system. Pressure perturbation is one method which can be applied to motile systems at different levels of organisation. We show here that pressure perturbs a specific interaction between actin and myosin in solution and also perturbs the cycling crossbridge in a contracting muscle.

The ability to exchange reversibly the regulatory light chains (RLCs) from scallop myosin has provided us with a test system to probe the mechanisms of regulation mediated by the RLCs from vertebrate skeletal, vertebrate smooth and molluscan myosins. The cloning and expression of these RLCs, together with domain-swapping and site-directed mutagenesis approaches, has allowed us to explore further the mechanisms involved and identify the functional importance of specific regions of the RLC molecule; for example, the presence of a high affinity metal binding site in the N-terminal domain and its interaction with the intact C-terminal domains are required for regulation.

Small angle X-ray scattering (SAXS) is a potentially powerful method for obtaining structural information from biological molecules in solution. The use of this technique in the laboratory has hitherto been limited by the long exposures necessary to obtain patterns on photographic film. Multi-wire area detectors, due to their high efficiency and absence of noise, enable patterns to be collected much more rapidly, typically in 1-2 h for a typical protein using laboratory sources. This opens up the possibility of using the technique on a semi-routine basis for a wide variety of problems. We outline the use of SAXS to characterise a large conformational change of myosin.

In this article we summarize our recent experiments studying the phosphorylation of vertebrate myosin heavy chains by protein kinase C and casein kinase II. Protein kinase C phosphorylates vertebrate non-muscle myosin heavy chains both in vitro and in intact cells. A single serine residue near the end of the helical portion of the myosin rod is the only site phosphorylated in a variety of vertebrate nonmuscle myosin heavy chains. There does not appear to be a site for protein kinase C phosphorylation in vertebrate smooth muscle myosin heavy chains. Casein kinase II phosphorylates a single serine residue located near the carboxyl terminus of the 204 x 10(3) Mr smooth muscle myosin heavy chain in vitro as well as in cultured smooth muscle cells. It does not phosphorylate the 200 x 10(3) Mr smooth muscle myosin heavy chain. However, the site is present in vertebrate nonmuscle myosin heavy chains. The 204 x 10(3) Mr myosin heavy chain of embryonic chicken gizzard smooth muscle is exceptional in not containing a site for casein kinase II phosphorylation.

Complementation of alternating zones of positive and negative charge in the myosin rod enables molecules to interact in a number of ways. This accounts for the complexity of the molecular organisation of thick filaments. However, directed mutagenesis of expressed LMM cDNA indicated that charge zone complementation is not a major driving force in myosin polymerisation. Instead, it probably serves to prevent unfavourable interaction geometries.

We describe an experimental approach to the use of genetics to study muscle contraction in Drosophila melanogaster. Mutations induced by in vitro mutagenesis are inserted into the genome of flies using P-element mediated transformation, permitting the effects of the mutant genes to be studied in vivo in the indirect flight muscles (IFMs). Details of how mechanical experiments can be performed on skinned IFMs, despite their small size, are provided. The effects of two in vitro actin mutations, G368E and E316K, are described. The problems of performing biochemical and biophysical experiments on the IFMs and their myofibrillar proteins are described, together with indications as to how these may be overcome.