Plant action: Multiple levels of regulation

This lecture is devoted to the relative contribution of various levels of regulation of the actin cytoskeleton functioning in the cell. Regulation at the levels of gene expression, mRNA and protein synthesis and stability, processes of actin polymerization/depolymerization and actin structures reorganization is briefly considered. Novel information about the pathways of signal transduction to the actin cytoskeleton with the involvement of Arp2/3 complex and RIC proteins is highlighted.     

Septins regulate border cell surface geometry,shape, and motility downstream of Rho in Drosophila

1. Download : Download high-res image (260KB)
2. Download : Download full-size image

     

Cell and molecular biology of bryophytes: ultimate limits to the resolution of phylogenetic problems

Ultrastructure, biochemistry and 5S rRNA sequences link tracheophytes, bryophytes and charalean green algae, but the precise interrelationships between these groups remain unclear. Further major clarification now awaits primary sequence data. These are also needed to determine directionality in possible evolutionary trends within the bryophytes, but are unlikely to overturn current schemes of classification or phylogeny. Comparative ultrastructural studies of spermatogenesis, sporogenesis, the cytoskeleton and plastids reinforce biochemical and morphogenetic evidence for the wide phyletic discontinuities between mosses, hepatics and hornworts, and also rule out direct lines of descent between them. Direct ancestral lineages from charalean algae to bryophytes and to tracheophytes are also unlikely. EM studies of gametophyte/sporophyte junctions, plus immunological investigations of bryophyte cytoskeletons, are likely to accentuate the differences between mosses, hepatirs and hornworts. Other priorities for systematics include elucidation of oil body ultrastructure, analysis of the changes in nuclear proteins during spermatogenesis and a detailed comparison of bryophyte and charalean plastids. The combined evidence from ultrastrueture, biochemistry, morphology and morphogenesis warrants general acceptance of the polyphyletic origin of the bryophytes. Ultrastructural attributes should be more widely used in bryophyte systematics.     

The structure of epidermal feet during their development

Epidermal cells in Calpodes and other insects form basal processes or feet that at first extend axially and later shorten at the same time as the larval segment shortens to the pupal shape. The feet grow into spaces at the surfaces of other cells to make a basal interlacing meshwork of cellular extensions that are combined mechanically by their desmosomal attachments to cell bodies above and to the basal lamina below. Microtubules and microfilaments are linked to these junctions by a reticular fibrous matrix. Gap junctions on the feet may couple cells that are several cell bodies removed from one another. The meshwork is also a sieve separating the hemolymph from the spaces between cells to form an intercellular compartment. Entry to the intercellular compartment is through the sieve made by the negatively charged basolateral cell surfaces that can prevent the entry of positively charged molecules such as cationic ferritin. As the cells become columnar, coincident with the metamorphic change in segment shape, the feet shorten and pack more densely together. At this time the basal lamina buckles axially as if responding to contraction of the feet. Segment shape change involves cell rearrangement and relative cell movement, necessitating the transient loss of plasma membrane plaque attachments to the cuticle apically and the loss of junctions laterally. Gap junctions involute in characteristic vacuoles. The metamorphic reduction in cell surface area coincides with the loss of basolateral membrane in smooth tubes and vesicles and the turnover of the apical surface in multivesicular bodies. New apical plasma membrane plaques and new lateral and basal junctions stabilize the cells in their pupal positions.     

Visualization of cytoskeletal changes through the life cycle inAcetabularia

Summary The cytoskeleton in the siphonous, marine green algaAcetabularia is visualized by immunocytochemistry using antibodies against plant alfa tubulin and animal smooth muscle actin. In the vegetative phase of the life cycle, when the cell grows a cylindrical stalk and until the reproductive cap is completed, actin forms continuous, parallel bundles that extend through the entire length of the stalk and cap rays respectively. Microtubules (MTs) cannot be detected until the primary nucleus, located in the rhizoid of the giant cell, divides to form thousands of secondary nuclei. MTs can then be seen radiating from each secondary nucleus that is encountered in the stalk on its migration upwards into the cap rays. They are oriented mostly parallel to the long axis of the cell. At arrival in the cap rays up to the white spot stage, when nuclei assume equidistant positions in the cap ray cytoplasm, a radiating system of MTs forms around each nucleus and dramatically increases until impressive radial arrays have developed. This phase coincides with a disappearance of actin bundles in the cap rays, but they are retained in the stalk cytoplasm. Shortly after that additional MTs appear around the disk like partitions of cap ray cytoplasm. Concomitantly, bundles of actin reappear colinearly with the circumferrential MTs eventually forming complete rings around each disk of cap ray cytoplasm. During this process the compartments of the future cysts are gradually bulging outwards and simultaneously the rings of actin sink inwards until domes are formed with the nuclei fixed in the top centers of the domes. At this stage the peripheral areas of the radiating MT systems around the nuclei start to break down, whereas the circumferrential MT systems remain intact. Subsequently, the rings of both actin and MTs decrease in diameter, and finally contract to a spot opposite the nucleus, while the cysts continue to develop their oval shape. After the cysts have become separated, they round up and enter several rounds of nuclear divisions. MTs form short radial arrays around each nucleus with minor changes due to a reduction of MTs during division followed by a reappearance after completion of each division. Actin is rearranged in the cysts to a cortical network of randomly oriented, short bundles, that is maintained until gamete formation sets in.These findings accentuate the involvement of Cytoskeletal elements in the key steps of morphogenesis inAcetabularia to an extent that is unknown in higher plants.     

Human arterial smooth muscle cells in culture: Inverse relationship between proliferation and expression of contractile proteins

Summary Human arterial smooth muscle cells (hASMC) from explants of the inner media of uterine arteries were studied in secondary culture. We had previously found that these cells depend on exogenous platelet-derived growth factor (PDGF) for proliferation in vitro. Deprivation of the serum mitogen(s) by culture in plasma-derived serum or bovine serum albumin (BSA) caused a true growth arrest that was reversible upon reexposure to the mitogen(s). When added to serum-containing medium, heparin caused a reversible growth arrest which could be competed for by increasing concentrations of serum. In the current study we used a set of smooth muscle-specific actin and myosin, antibodies to study the expression of contractile proteins in stress fibers under indirect immunofluorescence on hASMC in culture. Even in sparse culture, grwoth-arrested hASMC expressed stress fibers containing these actin and myosin epitopes. This was true irrespective of whether growth arrest was achieved by culture in media containing only BSA or a combination of heparin and whole blood serum. hASMC proliferating in whole blood serum in sparse culture did not express such strees fibers, as judged by immunofluorescent staining. This was true also for cells that were restimulated to proliferate in serum after a growth arrest. Utilizing a monoclonal antibody against a nuclear antigen expressed in proliferating human cells, we were able to demonstrate an inverse relationship between the expression of this antigen and the SMC-specific contractile proteins, respectively. Under these culture conditions, the reversible transition between defifferentiated and differentiated hASMC was almost complete and terminated about 1 wk after the change in culture condition. We conclude that hASMC in vitro respond, to exogenous PDGF by proliferation and dedifferetiation as a single population of cells. We also conclude that this modulation is reversible, because the cells become uniformly quiescent and differentiated when the mitogenic stimulus is blocked or removed. This study was supported by grants from the Swedish Medical Research Council (Project no. 4531 and 6816), the Swedish Association against Heart and Chest Diseases, the King Gustaf V and Queen Victoria Foundation, the National Institutes of Health, Bethesda, MD (grant HL 29873) and the Swedish National Board for Laboratory Animals.     

Effects of brain ischemia on intermediate filaments of rat hippocampus

Neurofilaments subunits (NF-H, NF-M, NF-L) and glial fibrillary acidic protein (GFAP) were investigated in the hippocampus of rats after distinct periods of reperfusion (1 to 15 days) following 20 min of transient global forebrain ischemia in the rat. In vitro [¹⁴Ca]leucine incorporation was not altered until 48 h after the ischemic insult, however concentration of intermediate filament subunits significantly decreased in this period. Three days after the insult, leucine incorporation significantly increased while the concentration NF-H, NF-M, and NF-L were still diminished after 15 days of reperfusion. In vitro incorporation of³²P into NF-M and NF-L suffered immediately after ischemia, but returned to control values after two days of reperfusion. GFAP levels decreased immediately after ischemia but quickly recovered and significantly peaked from 7 to 10 days after the insult. These results suggest that transient ischemia followed by reperfusion causes proteolysis of intermediate filaments in the hippocampus, and that proteolysis could be facilitated by diminished phosphorylation levels of NF-M and NF-L.     

Immunodetection of cytoskeletal structures and the Eg5 motor protein on deep-etch replicas of Xenopus egg cortices isolated during the cortical rotation

Summary— We have developed a new method for immunogold detection on deep-etch replicas of isolated Xenopus egg cortices in order to examine the interactions of different cortical elements in three dimensions at high resolution. We have applied this technique to vegetal cortices isolated during the second half of the first cell cycle. The vegetal cortical region at this time is the site of cellular machinery responsible for the ‘cortical rotation’. The entire cortex translocates with respect to the inner cytoplasm, relocating dorsalising determinants to the future dorsal side of the egg. The aligned microtubules in the shear zone between cytoplasm and cortex, implicated in the cortical rotation, were found to be organised as interweaving loose bundles. Interleaved amongst these aligned microtubules were extensive sheets of ER lying in layers parallel to the egg surface. Cytokeratin filaments were found to associate closely with the microtubules over short stretches. Putative actin filaments were present in the shear zone and in the cortex. Eg5, an abundant kinesin-related microtubule motor protein, and candidate for a role in generating cortical rotation movement, showed an almost exclusive localisation to microtubules. Immunofluorescence studies of cortices treated with detergent to disrupt ER or cold to depolymerise microtubules confirmed that Eg5 associates primarily with microtubules. We propose revised models for the mechanism of cortical rotation based on these observations and conclude that Eg5 is unlikely to move ER relative to microtubules during the cortical rotation.     

Function of osteocytes in bone

Although the structural design of cellular bone (i.e., bone containing osteocytes that are regularly spaced throughout the bone matrix) dates back to the first occurrence of bone as a tissue in evolution, and although osteocytes represent the most abundant cell type of bone, we know as yet little about the role of the osteocyte in bone metabolism. Osteocytes descend from osteoblasts. They are formed by the incorporation of osteoblasts into the bone matrix. Osteocytes remain in contact with each other and with cells on the bone surface via gap junction–coupled cell processes passing through the matrix via small channels, the canaliculi, that connect the cell body–containing lacunae with each other and with the outside world. During differentiation from osteoblast to mature osteocyte the cells lose a large part of their cell organelles. Their cell processes are packed with microfilaments. In this review we discuss the various theories on osteocyte function that have taken in consideration these special features of osteocytes. These are (1) osteocytes are actively involved in bone turnover; (2) the osteocyte network is through its large cell-matrix contact surface involved in ion exchange; and (3) osteocytes are the mechanosensory cells of bone and play a pivotal role in functional adaptation of bone. In our opinion, especially the last theory offers an exciting concept for which some biomechanical, biochemical, and cell biological evidence is already available and which fully warrants further investigations. © 1994 Wiley-Liss, Inc.