Induction of cortical oscillations in spreading cells by depolymerization of microtubules.

Actomyosin-based cortical contractility is a common feature of eukaryotic cells but the capability to produce rhythmic contractions is found in only a few types such as cardiomyocytes. Mechanisms responsible for the acquisition of this capability remain largely unknown. Rhythmic contractility can be induced in non-muscle cells by microtubule depolymerization. Spreading epithelial cells and fibroblasts in which microtubules were depolymerized with nocodazole or colcemid underwent rhythmic oscillations of the body that lasted for several hours before the cells acquired a stable, flattened shape. By contrast, control cells spread and flattened into discoid shapes in a smooth and regular manner. Quantitative analysis of the oscillations showed that they have a period of about 50 seconds. The kinase inhibitors, HA 1077 and H7, and the more specific rho-kinase inhibitor, Y 27632, caused the oscillations to immediately cease and the cells to become flat. Transient increases in cytoplasmic calcium preceded the contractile phase of the oscillations. Wrinkle formation by cells plated on elastic substrata indicated that the contractility of colcemid-treated cells increased in comparison to controls but was drastically decreased after HA 1077 addition. These data suggest that an intact microtubular system normally prevents pulsations by moderating excessive rho-mediated actin myosin contractility. Possible mechanistic interactions between rho-mediated and calcium activated contractile pathways that could produce morphological oscillations are discussed.

Tails of unconventional myosins.

In addition to the conventional myosins (class II) required for processes such as muscle contraction and cytokinesis, the myosin superfamily of actin-based motor proteins includes at least 14 'unconventional' classes. These unconventional myosins are defined by myosin-like head (motor) domains attached to class-specific tail domains that differ greatly from those of myosin-II. The unconventional myosins account for almost two-thirds of the 28 or more myosin genes currently believed to be expressed in humans and 80-90% of the approximately 10 or more myosin genes expressed in a typical nonmuscle cell. Although these members of the myosin superfamily have not been as intensively investigated as the conventional myosins, unconventional myosins are known or believed to power many forms of actin-based motility and organelle trafficking. The presence of signaling domains such as kinase domains, SH3 domains, PH domains or GTPase-activating domains in the tails of unconventional myosins indicates that these proteins can also be components of signal transduction pathways. Since several classes of the myosin superfamily have been found only in lower eukaryotes or plants (VIII, XI, XIII and XIV), in this review we will focus on the structures and properties of the unconventional myosins found in multicellular animals (excluding classes I and V, which have been reviewed elsewhere recently). Special attention will be focused on the three classes of unconventional myosins that can cause deafness in mouse or humans when mutated. In addition, we discuss the discovery of a pair of intriguing domains, the Myosin Tail Homology 4 (MyTH4) and FERM (band 4.1, Ezrin, Radixin, Moesin) domains, that are present in the tails of otherwise very different myosins as well as a plant kinesin-like protein. Recent progress in the identification of novel unconventional myosins will also be summarized.

Colloid osmotic pressure of steer alpha- and beta-crystallins: possible functional roles for lens crystallin distribution and structural diversity.

This study addresses the general mechanisms whereby the major cytoplasmic proteins from the adult bovine lens contribute both to transparency and maintenance of the refractive index gradient across the lens. Colloid osmotic properties and quaternary structure were measured for alpha- and beta-crystallins isolated from the steer lens, including low-molecular-weight crystallins from the cortex (alpha Le and beta L) and nucleus (alpha Ln) and high-molecular-weight crystallins from the nucleus (alpha H and beta H). In electron microscopic images of rotary-shadowed preparations alpha Le appears as spherical particles 16 nm in diameter, alpha Ln appeared as individual spheres or small aggregates of spherical subunits, alpha H contained large irregular aggregates as large as 180 nm, and both beta L and beta H appeared as elliptical particles of 7-9 nm diameter. Secondary osmometry showed that for all these crystallins colloid osmotic pressure increased monotonically in a non-linear fashion with protein concentration. For the alpha-crystallins, osmotic pressure rose more steeply with concentration for alpha Le than for either alpha Ln or alpha H, so that at 0.3 g ml-1 at 0.1 M ionic strength, the colloid osmotic pressure of alpha Le, alpha Ln and alpha H were approximately 2.6 x 10(5) dyn cm-2, 1.6 x 10(5) dyn cm-2 and 1.0 x 10(5) dyn cm-2, respectively. In a similar manner, osmotic pressure rose more steeply with concentration of beta L than for beta H, so that at 0.3 g ml-1 at 0.1 M ionic strength the colloid osmotic pressures of beta L and beta H were 2.6 x 10(5) dyn cm-2 and 1.1 x 10(5) dyn cm-2, respectively. The osmotic pressure of alpha Le dropped as ionic strength was increased from 0.02 to 0.4 M. For beta L and beta H, osmotic pressure dropped as ionic strength was increased from 0.02 to 0.1 M but was nearly the same at 0.1 M and 0.4 M ionic strength. The data for steer alpha Ln and beta H were similar to previous reports for calf cortical alpha L and beta-crystallins, respectively. The osmotic pressure isotherms for alpha Le, beta L and that previously reported for steer cortical extract were nearly identical, whereas the nuclear crystallins (alpha Ln, alpha H or beta H) generated slightly higher pressures than those previously reported for steer nuclear crystallin extracts. In all cases, osmotic pressure rose more steeply with concentration for the cortical crystallins than for the nuclear crystallins.(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular architecture in age-related human nuclear cataracts.

Age-related or senile human nuclear cataracts were examined using electron microscopy of thin sections prepared from thick vibrating-knife microtome sections of nuclei extracted by extracapsular surgery. The use of extended aldehyde-tannic acid fixation of 80-120-microns thick vibrating-knife microtome sections overcame the difficult problem of preserving the hardened nuclear core of aged lenses. Comparisons were made between a typical nuclear cataract, containing a central opacity and a transparent rim, and a more advanced, or mature, completely opaque nuclear cataract. The typical nuclear cataract contained no obvious cell disruption, cellular debris, or objects that readily could explain the central opacity. The fiber cells had intact uniformly stained cytoplasms with well-defined plasma membrane borders and gap junctions. The transparent rim and the nuclear core appeared similar, except that fiber cells in the nucleus were more condensed with more elaborate intercellular interdigitations. The mature cataract showed various types of cell disruption in the perimeter but not in the core of the nucleus. These disruptions were globules, vacuoles, multilamellar membranes, and clusters of highly undulating membranes. Because these potential scattering centers were not found in the nuclear core, they probably were not the sole cause of the observed opacity. Other potential scattering centers found throughout the mature cataract nucleus included variations in staining density between adjacent cells, enlarged extracellular spaces between undulating membrane pairs, and protein-like deposits in the extracellular space. Similar features, although less pronounced, were present in the typical nuclear cataract. It was concluded that massive cell disruption is not essential to the formation of a central nuclear opacity. Subtle structural changes, especially small fluctuations in protein density between adjacent cells and alterations of the membranes and the extracellular space, probably contribute significantly to the central opacities in human nuclear cataracts.

The distribution and assessment of electron-microscopic abnormalities of human cilia.

The aim of this study was to overcome difficulties of assessing the true incidence of electromicroscopic abnormalities of microtubular structure of cilia by examining large numbers of cilia from each case. The effects of different fixatives on the appearances of cilia were also studied. Bronchial biopsies were examined from 35 subjects who were being investigated for various lung diseases and nasal biopsies from 12 subjects (7 with retinitis pigmentosa (R.P.), and 5 healthy controls). Numerous pieces of normal looking bronchial wall from a lobectomy specimen were used to examine the effect of six different fixatives. 2.9% of bronchial cilia (mean of 890 cilia examined) and 2.4% of nasal cilia (mean of 808 cilia examined) showed microtubular abnormalities. Examining large numbers of cilia established that increased microtubular abnormalities were associated with smoking, chronic pulmonary infection and carcinoma of the lung. There was a significant increase (p less than 0.001) in microtubular abnormalities in nasal cilia in R.P. The appearances of cilia varied considerably with different fixatives. The numbers of dynein arms seen and the ease of recognising radial spokes and microtubules was particularly effected by fixation. The true incidence of microtubular abnormalities can only be ascertained by examining large numbers of cilia.