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NASA NTRS1992

Plastids: dynamic components of plant cell development

The gravitropic bending of maize roots, as a response to reorientation of the root within a gravitational field, was examined for sensitivity to exogenous applications of the cytoskeletal inhibitor, cytochalasin D. Agar blocks were impregnated with this inhibitor, and were applied either to the root cap or to the zone of root cell elongation. Root growth was normal with either treatment, if the roots were not repositioned with respect to the gravitational vector. When untreated roots were placed in a horizontal position with respect to gravity, a 40 degree bending response was observed within one hour. This bending also occurred when cytochalasin D was applied at high concentrations to the zone of root cell elongation. However, when cytochalasin D above 40 micrograms/ml was applied to the root cap, roots lost the ability of directional reorientation within the gravitational field, causing a random bending.

NASA Discipline Cell Biology↗

NASA NTRS1997

Cytochalasin D does not inhibit gravitropism in roots

It is generally thought that sedimenting plastids are responsible for gravity sensing in higher plants. We directly tested the model generated by the current statolith hypothesis that the gravity sensing that leads to gravitropism results from an interaction between the plastids and actin microfilaments. We find that the primary roots of rice, corn, and cress undergo normal gravitropism and growth even when exposed to cytochalasin D, a disruptor of actin microfilaments. These results indicate that an interaction between amyloplasts and the actin cytoskeleton is not critical for gravity sensing in higher plants and weaken the current statolith hypothesis.

NASA Discipline Plant Biology↗

NASA NTRS1995

Effects of clinorotation and microgravity on sweet clover columella cells treated with cytochalasin D

The cytoskeleton of columella cells is believed to be involved in maintaining the developmental polarity of cells observed as a reproducible positioning of cellular organelles. It is also implicated in the transduction of gravitropic signals. Roots of sweet clover (Melilotus alba L.) seedlings were treated with a microfilament disrupter, cytochalasin D, on a slowly rotating horizontal clinostat (2 rpm). Electron micrographs of treated columella cells revealed several ultrastructural effects including repositioning of the nucleus and the amyloplasts and the formation of endoplasmic reticulum (ER) whorls. However, experiments performed during fast clinorotation (55 rpm) showed an accumulation (but no whorling) of a disorganized ER network at the proximal and distal pole and a random distribution of the amyloplasts. Therefore, formation of whorls depends upon the speed of clinorotation, and the overall impact of cytochalasin D suggests the necessity of microfilaments in organelle positioning. Interestingly, a similar drug treatment performed in microgravity aboard the US Space Shuttle Endeavour (STS-54, January 1993) caused a displacement of ER membranes and amyloplasts away from the distal plasma membrane. In the present study, we discuss the role of microfilaments in maintaining columella cell polarity and the utility of clinostats to simulate microgravity.

manned↗

NASA NTRS2001

Regulation of PGE(2) and PGI(2) release from human umbilical vein endothelial cells by actin cytoskeleton

Disruption of microfilaments in human umbilical vein endothelial cells (HUVEC) with cytochalasin D (cytD) or latrunculin A (latA) resulted in a 3.3- to 5.7-fold increase in total synthesis of prostaglandin E(2) (PGE(2)) and a 3.4- to 6.5-fold increase in prostacyclin (PGI(2)) compared with control cells. Disruption of the microtubule network with nocodazole or colchicine increased synthesis of PGE(2) 1.7- to 1.9-fold and PGI(2) 1.9- to 2.0-fold compared with control cells. Interestingly, however, increased release of PGE(2) and PGI(2) from HUVEC into the media occurred only when microfilaments were disrupted. CytD treatment resulted in 6.7-fold more PGE(2) and 3.8-fold more PGI(2) released from HUVEC compared with control cells; latA treatment resulted in 17.7-fold more PGE(2) and 11.2-fold more PGI(2) released compared with control cells. Both increased synthesis and release of prostaglandins in response to all drug treatments were completely inhibited by NS-398, a specific inhibitor of cyclooxygenase-2 (COX-2). Disruption of either microfilaments using cytD or latA or of microtubules using nocodazole or colchicine resulted in a significant increase in COX-2 protein levels, suggesting that the increased synthesis of prostaglandins in response to drug treatments may result from increased activity of COX-2. These results, together with studies demonstrating a vasoprotective role for prostaglandins, suggest that the cytoskeleton plays an important role in maintenance of endothelial barrier function by regulating prostaglandin synthesis and release from HUVEC.

Non-NASA Center↗

NASA NTRS2002

Spatial and temporal traction response in human airway smooth muscle cells

Tractions that cells exert on their substrates are essential in cell spreading, migration, and contraction. These tractions can be determined by plating the cells on a flexible gel and measuring the deformation of the gel by using fluorescent beads embedded just below the surface of the gel. In this article we describe the image correlation method (ICM) optimized for determining the displacement field of the gel under a contracting cell. For the calculation of the traction field from the displacement field we use the recently developed method of Fourier transform traction cytometry (FTTC). The ICM and FTTC methods are applied to human airway smooth muscle cells during stimulation with the contractile agonist histamine or the relaxing agonist isoproterenol. The overall intensity of the cell contraction (the median traction magnitude, the energy transferred from the cell to the gel, and the net contractile moment) increased after activation with histamine, and decreased after treatment with isoproterenol. Cells exhibited regional differences in the time course of traction during the treatment. Both temporal evolution and magnitude of traction increase induced by histamine varied markedly among different cell protrusions, whereas the nuclear region showed the smallest response. These results suggest that intracellular mediators of cell adhesion and contraction respond to contractile stimuli with different rates and intensities in different regions of the cell.

NASA Discipline Cell Biology↗

NASA NTRS2000

Maintenance of asymmetric cellular localization of an auxin transport protein through interaction with the actin cytoskeleton

In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.

Non-NASA Center↗

NASA NTRS1995

Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis

Capillary endothelial cells can be switched between growth and differentiation by altering cell-extracellular matrix interactions and thereby, modulating cell shape. Studies were carried out to determine when cell shape exerts its growth-regulatory influence during cell cycle progression and to explore the role of cytoskeletal structure and mechanics in this control mechanism. When G0-synchronized cells were cultured in basic fibroblast growth factor (FGF)-containing defined medium on dishes coated with increasing densities of fibronectin or a synthetic integrin ligand (RGD-containing peptide), cell spreading, nuclear extension, and DNA synthesis all increased in parallel. To determine the minimum time cells must be adherent and spread on extracellular matrix (ECM) to gain entry into S phase, cells were removed with trypsin or induced to retract using cytochalasin D at different times after plating. Both approaches revealed that cells must remain extended for approximately 12-15 h and hence, most of G1, in order to enter S phase. After this restriction point was passed, normally 'anchorage-dependent' endothelial cells turned on DNA synthesis even when round and in suspension. The importance of actin-containing microfilaments in shape-dependent growth control was confirmed by culturing cells in the presence of cytochalasin D (25-1000 ng ml-1): dose-dependent inhibition of cell spreading, nuclear extension, and DNA synthesis resulted. In contrast, induction of microtubule disassembly using nocodazole had little effect on cell or nuclear spreading and only partially inhibited DNA synthesis. Interestingly, combination of nocodazole with a suboptimal dose of cytochalasin D (100 ng ml-1) resulted in potent inhibition of both spreading and growth, suggesting that microtubules are redundant structural elements which can provide critical load-bearing functions when microfilaments are partially compromised. Similar synergism between nocodazole and cytochalasin D was observed when cytoskeletal stiffness was measured directly in living cells using magnetic twisting cytometry. These results emphasize the importance of matrix-dependent changes in cell and nuclear shape as well as higher order structural interactions between different cytoskeletal filament systems for control of capillary cell growth during angiogenesis.

Non-NASA Center↗

NASA NTRS1993

Cytoskeleton-amyloplast interactions in sweet clover

The distribution of organelles within columella cells of sweet clover was examined by transmission electron microscopy following growth under static or clinorotating conditions. A developmentally conditioned polarity was observed, with a proximal location of the nucleus and a distal accumulation of the endoplasmic reticulum. This polarity was insensitive to clinorotation. In contrast, clinorotation altered the location of amyloplasts. Application of cytoskeletal poisons (colchicine, cytochalasin D, taxol, and phalloidin), especially during clinorotation, had interesting effects on the maintenance of columella cell polarity, with a profound effect on the extent, location, and structure of the endoplasmic reticulum. The site of cytoskeletal interactions with sedimenting amyloplasts is thought to be the amyloplast envelope. An envelope fraction, having over 17 polypeptides, was isolated using immobilized antibody technology, and will provide a means of assessing the role of specific peptides in cytoskeleton/amyloplast interactions.

Non-NASA Center↗

NASA NTRS1997

The organization of the actin cytoskeleton in vertical and graviresponding primary roots of maize

To determine whether actin microfilament (MF) organization is correlated with differential elongation, primary roots of Zea mays cv Merit maintained vertically or reoriented horizontally for 15 to 120 min were stained with rhodamine phalloidin and examined with a confocal microscope. Root curvature was measured with a computer-controlled video digitizer. In vertical roots bundles of MFs in the elongation and maturation zone were oriented parallel to the longitudinal axis of cells. MFs in the vascular parenchyma cells were more abundant than in the cortex and epidermis. Epidermal and proendodermal cells in the meristematic region contained transverse cortical MFs. The organization of MFs of graviresponding roots was similar to that of vertical roots. Application of cytochalasin B or cytochalasin D resulted in extensive disruption of MFs in the cortex and epidermis, but only partially affected MFs in the stele. Despite the cytochalasin B-induced depolymerization of MFs, gravicurvature exceeded that of controls. In contrast, the auxin transport inhibitor N-1 naphthylphthalamic acid suppressed root curvature but had no observable effect on the integrity of the MFs. The data indicate that MFs may not be involved in the graviresponse of maize roots.

NASA Discipline Plant Biology↗

NASA NTRS1990

Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly and bone resorption

The mechanisms of Ca2+ entry and their effects on cell function were investigated in cultured chicken osteoclasts and putative osteoclasts produced by fusion of mononuclear cell precursors. Voltage-gated Ca2+ channels (VGCC) were detected by the effects of membrane depolarization with K+, BAY K 8644, and dihydropyridine antagonists. K+ produced dose-dependent increases of cytosolic calcium ([Ca2+]i) in osteoclasts on glass coverslips. Half-maximal effects were achieved at 70 mM K+. The effects of K+ were completely inhibited by dihydropyridine derivative Ca2+ channel blocking agents. BAY K 8644 (5 X 10(-6) M), a VGCC agonist, stimulated Ca2+ entry which was inhibited by nicardipine. VGCCs were inactivated by the attachment of osteoclasts to bone, indicating a rapid phenotypic change in Ca2+ entry mechanisms associated with adhesion of osteoclasts to their resorption substrate. Increasing extracellular Ca2+ ([Ca2+]e) induced Ca2+ release from intracellular stores and Ca2+ influx. The Ca2+ release was blocked by dantrolene (10(-5) M), and the influx by La3+. The effects of [Ca2+]e on [Ca2+]i suggests the presence of a Ca2+ receptor on the osteoclast cell membrane that could be coupled to mechanisms regulating cell function. Expression of the [Ca2+]e effect on [Ca2+]i was similar in the presence or absence of bone matrix substrate. Each of the mechanisms producing increases in [Ca2+]i, (membrane depolarization, BAY K 8644, and [Ca2+]e) reduced expression of the osteoclast-specific adhesion structure, the podosome. The decrease in podosome expression was mirrored by a 50% decrease in bone resorptive activity. Thus, stimulated increases of osteoclast [Ca2+]i lead to cytoskeletal changes affecting cell adhesion and decreasing bone resorptive activity.

NASA Discipline Musculoskeletal↗