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1.
When neurons swell and shrink they extensively reorganize their plasma membrane. A striking aspect of these membrane dynamics
is the transient appearance of vacuole-like dilations (VLDs) which, counterintuitively, expand as the neurons shrink. Here,
confocal microscopy of cultured molluscan (Lymnaea) neurons was used in conjunction with aqueous phase and membrane dyes to examine changing VLD membrane topology as VLDs form,
reverse or recover. We show that VLDs start as discrete invaginations at the adherent surface, so VLD and plasma membranes
are initially contiguous. Over the next few minutes VLDs expand and penetrate the cytoplasm. At the substratum, the mouths
of VLDs develop into irregular annuli of motile adherent processes whereas deeper in the cytoplasm, VLD membrane profiles
are smooth. Subsequently VLDs spontaneously shrink; as this recovery proceeds, constriction of the motile VLD mouth leads
to the internalization of plasma membrane. Washout experiments with aqueous phase dyes demonstrated that VLD constriction
yields bona fide vacuoles, i.e., membrane-bound compartments isolated from the external medium. VLDs can also be experimentally eliminated
by returning cells to swelling conditions; this reversal process drives membrane back to the surface.
VLD formation and reinternalization of VLD membrane can be seen as aspects of plasma membrane surface area regulation. We
postulate that area adjustments, driven by regional membrane tension differences, become noticeable when excessive perturbations
overload normal membrane reprocessing steps. Both the changes in VLD membrane topology, and previously established capacitance
changes accompanying cell shrinking and swelling, argue that osmomechanically perturbed neurons regulate their surface area
as their volume changes.
Received: 13 May 1998/Revised: 18 September 1998 相似文献
2.
T.L. Herring C.S. Cohan E.A. Welnhofer L.R. Mills C.E. Morris 《The Journal of membrane biology》1999,171(2):151-169
Neuronal shape and volume changes require accompanying cell surface adjustments. In response to osmotic perturbations, neurons
show evidence of surface area regulation; shrinking neurons invaginate membrane at the substratum, pinch off vacuoles, and
lower their membrane capacitance. F-actin is implicated in reprocessing newly invaginated membrane because cytochalasin causes
the transient shrinking-induced invaginations, vacuole-like dilations (VLDs), to persist indefinitely instead of undergoing
recovery. To help determine if cortical F-actin indeed contributes to cell surface area regulation, we test, here, the following
hypothesis: invaginating VLD membrane rapidly establishes an association with F-actin and this association contributes to
VLD recovery. Cultured molluscan (Lymnaea) neurons, whose large size facilitates three-dimensional imaging, were used. In fixed neurons, fluorescent F-actin stains
were imaged. In live neurons, VLD membrane was monitored by brightfield microscopies and actin was monitored via a fluorescent
tag. VLD formation (unlike VLD recovery) is cytochalasin insensitive and consistent with this, VLDs formed readily in cytochalasin-treated
neurons but showed no association with F-actin. Normally, however (i.e., no cytochalasin), VLDs were foci for rapid reorganization
of F-actin. At earliest detection (1–2 min), nascent VLDs were entirely coated with F-actin and by 5 min, VLD mouths (i.e.,
at the substratum) had become annuli of F-actin-rich motile leading edge. Time lapse images from live neurons showed these
rings to be motile filopodia and lamellipodia. The retrieval of VLD membrane (vacuolization) occurred via actin-associated
constriction of VLD mouths. The interplay of surface membrane and cortical cytoskeleton in osmotically perturbed neurons suggests
that cell surface area and volume adjustments are coordinated in part via mechanosensitive F-actin dynamics.
Received: 25 March 1999/Revised: 15 June 1999 相似文献
3.
Nicolas Groulx Francis Boudreault Sergei N. Orlov Ryszard Grygorczyk 《The Journal of membrane biology》2006,214(1-2):43-56
To accommodate expanding volume (V) during hyposmotic swelling, animal cells change their shape and increase surface area
(SA) by drawing extra membrane from surface and intracellular reserves. The relative contributions of these processes, sources
and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o−, CHO and NIH 3T3 cells were evaluated by reconstructing cell three-dimensional topology based on conventional light microscopic
images acquired simultaneously from two perpendicular directions. The size of SA reserves was determined by swelling cells
in extreme 98% hypotonic (∼6 mOsm) solution until membrane rupture; all cell types examined demonstrated surprisingly large
membrane reserves and could increase their SA 3.6 ± 0.2-fold and V 10.7 ± 1.5-fold. Blocking exocytosis (by N-ethylmaleimide or 10°C) reduced SA and V increases of A549 cells to 1.7 ± 0.3-fold and 4.4 ± 0.9-fold, respectively. Interestingly,
blocking exocytosis did not affect SA and V changes during moderate swelling in 50% hypotonicity. Thus, mammalian cells accommodate
moderate (<2-fold) V increases mainly by shape changes and by drawing membrane from preexisting surface reserves, while significant
endomembrane insertion is observed only during extreme swelling. Large membrane reserves may provide a simple mechanism to
maintain membrane tension below the lytic level during various cellular processes or acute mechanical perturbations and may
explain the difficulty in activating mechanogated channels in mammalian cells.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
4.
Membrane reserves and hypotonic cell swelling 总被引:2,自引:0,他引:2
To accommodate expanding volume (V) during hyposmotic swelling, animal cells change their shape and increase surface area (SA) by drawing extra membrane from surface and intracellular reserves. The relative contributions of these processes, sources and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o(-), CHO and NIH 3T3 cells were evaluated by reconstructing cell three-dimensional topology based on conventional light microscopic images acquired simultaneously from two perpendicular directions. The size of SA reserves was determined by swelling cells in extreme 98% hypotonic (approximately 6 mOsm) solution until membrane rupture; all cell types examined demonstrated surprisingly large membrane reserves and could increase their SA 3.6 +/- 0.2-fold and V 10.7 +/- 1.5-fold. Blocking exocytosis (by N-ethylmaleimide or 10 degrees C) reduced SA and V increases of A549 cells to 1.7 +/- 0.3-fold and 4.4 +/- 0.9-fold, respectively. Interestingly, blocking exocytosis did not affect SA and V changes during moderate swelling in 50% hypotonicity. Thus, mammalian cells accommodate moderate (<2-fold) V increases mainly by shape changes and by drawing membrane from preexisting surface reserves, while significant endomembrane insertion is observed only during extreme swelling. Large membrane reserves may provide a simple mechanism to maintain membrane tension below the lytic level during various cellular processes or acute mechanical perturbations and may explain the difficulty in activating mechanogated channels in mammalian cells. 相似文献
5.
Photofrin II is a photosensitizer frequently applied in photodynamic therapy. Light-induced tumor cell inactivation observed
in the presence of this substance has been suggested to start with modifications at the level of cellular membranes. In the
present study electrophysiological techniques are applied in order to investigate the action of photofrin II on functional
properties of the plasma membrane of opossum kidney (OK) cells (as an epithelial model system) and of fibroblasts. Illumination
of the cells in the presence of photofrin II (or Zn-phthalocyanine) leads to comparatively fast depolarization of the membrane
potential. It is caused by a strong change of the membrane conductance which proceeds in two phases. Both phases contribute
to a loss of ion selectivity of the plasma membrane between K+ and Na+. In the first phase, specific pathways for K+, which determine the resting potential under physiological conditions, are inactivated. The second phase is distinguished
by a marked increase of a nonselective conductance. The increase of the latter — after light-induced initiation — continues
in the dark. The conclusions are derived from light-induced, time-dependent changes of the membrane conductance and of the
shape of the current-voltage relationship detected under different experimental conditions.
Received: 26 May 1998/Revised: 8 September 1998 相似文献
6.
Summary. During stomatal movement, guard cells undergo large and reversible changes in cell volume and consequently surface area. These
alterations in surface area require addition and removal of plasma membrane material. How this is achieved is largely unknown.
Here we summarize recent studies of membrane turnover in guard cells using electrophysiology and fluorescent imaging techniques.
The results implicate that membrane turnover in guard cells and most likely in plant cells in general is sensitive to changes
in membrane tension. We suggest that this provides a mechanism for the adaptation of surface area of guard cells to osmotically
driven changes in cell volume. In addition, guard cells also exhibit constitutive membrane turnover. Constitutive and pressure-driven
membrane turnover were found to be associated with addition and removal of K+ channels. This implies that some of the exo- and endocytic vesicles carry K+ channels. Together the results demonstrate that exo- and endocytosis is an essential process in guard cell functioning.
Correspondence and reprints: Institute of Botany, Darmstadt University of Technology, Schnittspahnstrasse 3, 64287 Darmstadt,
Federal Republic of Germany. 相似文献
7.
M.E. Forero M. Marín A. Corrales I. Llano H. Moreno M. Camacho 《The Journal of membrane biology》1999,170(2):173-180
Whole cell patch-clamp recordings were used to study the electrical properties of the macrophage-like cell line J774.1, after
infection with Leishmania amazonensis. Infection induced a significant increase in cell size and membrane capacitance, suggesting that parasite invasion leads to
the addition of plasma membrane to the host cell. By 24 hr after infection, the host cell membrane potential was significantly
more hyperpolarized than control cells, and this difference remained for the subsequent 72 hr post-infection. The hyperpolarization
was paralleled by an increase in the density of inward rectifying K+ currents. The shape of the conductance vs. voltage curve, the kinetic properties and the pharmacological profile of these currents were not significantly altered by
infection. These results suggest that infection by L. amazonensis causes an increase in the number of functional inward rectifying K+ channels, leading to hyperpolarization of the host cell membrane.
Received: 19 January 1999/Revised: 20 April 1999 相似文献
8.
In this study, electrorotation spectra of individual cells (that is, frequency dependence of cell rotation speed) have been
proved to yield information not only about the passive electric properties of cell constituents, but also about the presence
of mobile charges within the plasma membrane being part of ion carrier transport systems. Experiments on human erythrocytes
pretreated with the lipophilic anion dipicrylamine (DPA) gave convincing evidence that these artificial mobile charges adsorbed
to the plasma membrane contributed significantly to the rotational spectrum at relatively low conductivity of the external
medium (2–5 mS m−1). Theoretical integration of the mobile charge concept into the single-shell model (viewing the cell as a homogenous sphere
surrounded by a membrane) led to a set of equations which predicted electrorotational behavior of DPA-treated cells in dependence
on medium conductivity. The quantitative data on the partition and the transmembrane translocation rate of the DPA anion extracted
from the experimental rotational spectra agreed well with the corresponding literature values.
Received: 14 February 1996/Revised: 29 May 1996 相似文献
9.
Partners in Protection: Interdependence of Cytoskeleton and Plasma Membrane in Adaptations to Applied Forces 总被引:5,自引:0,他引:5
In mechanically active environments mammalian cells must cope with potentially injurious forces to survive, but the most
proximal mechanosensors are largely unknown. How mechanoprotective responses to applied forces are generated and regulated
is still a mystery. We consider recent evidence that suggests cellular mechanoprotective adaptations involve a coordinated
remodeling of the cell membrane and the associated cytoskeleton. The plasma membrane ``protects' the cytoskeleton by maintenance
of intracellular ionic balance and can modulate force-induced cytoskeletal rearrangements by stretch-activated (e.g., Ca2+) ion channels and mechanosensitive enzymes (e.g., Phospholipase A2 and Phospholipase C). Conversely, the cytoskeleton protects the plasma membrane by providing structural support, reinforcement
of the cortical framework at sites of force application, modulation of mechanosensitive ion channels and by potentially contributing
to the membrane resealing process after mechanical rupture. We suggest that the plasma membrane and the cytoskeleton are partners
in the cytoprotective response to physical forces.
Received: 8 September 1999/Revised: 15 December 1999 相似文献
10.
The putative role of lysophospholipids in activation and regulation of the volume-sensitive taurine efflux was investigated
in HeLa cells using tracer technique. Lysophosphatidylcholine (LPC, 10 μm) with oleic acid increased taurine efflux during hypotonic and isotonic conditions. Substituting palmitic or stearic acid
for oleic acid enhanced taurine release during isotonic conditions, whereas ethanolamine, serine or inositol containing lysophospholipids
were ineffective. High concentrations of LPC (25 μm) induced Ca2+ influx, loss of adenosine nucleotides, taurine and the Ca2+-sensitive probe Fura-2, and thus reflected a general breakdown of the membrane permeability barrier. Low concentrations of
LPC (5–10 μm) solely induced taurine efflux. The LPC-induced taurine release was unaffected by anion channel blockers (DIDS, MK196) and
the 5-lipoxygenase inhibitor ETH 615-139, which all blocked the volume sensitive taurine efflux. Furthermore, LPC-induced
taurine release was reduced by antioxidants (NDGA, vitamin E) and the protein tyrosine kinase inhibitor genistein. The swelling-induced
taurine efflux was in the absence of LPC unaffected by vitamin E, blocked by genistein, and increased by H2O2 and the protein tyrosine phosphatase inhibitor vanadate. It is suggested that low concentrations of LPC permeabilizes the
plasma membrane in a Ca2+-independent process that involves generation of reactive oxygen species and tyrosine phosphorylation, and that LPC is not
a second messenger in activation of the volume sensitive taurine efflux in HeLa cells.
Received: 17 December 1999/Revised: 13 April 2000 相似文献
11.
M.G. Leonardi P. Parenti M. Casartelli B. Giordana 《The Journal of membrane biology》1997,159(3):209-217
The mechanical properties of brush border membrane vesicles, BBMV, from rabbit kidney proximal tubule cells, were studied
by measuring the initial and final equilibrium volumes of vesicles subjected to different osmotic shocks, using cellobiose
as the impermeant solute in the preparation buffer.
An elevated intracellular hydrostatic pressure was inferred from osmotic balance requirements in dilute solutions. For vesicles
prepared in 18 and 85 mosm solutions, these pressures are close to 17 mosm (290 mm Hg). The corresponding membrane surface tension is 6.0 × 10−5 N cm−1 while the membrane surface area is expanded by at least 2.2%. When these vesicles are exposed to very dilute solutions the
internal hydrostatic pressure rises to an estimated 84 mosm (1444 mm Hg) just prior to lysis. The corresponding maximal surface tension (pre-lysis) is 18.7 × 10−5 N cm−1, and the maximal expansion of membrane area is 6.8%. The calculated area compressibility elastic modulus was 2.8 × 10−3 N cm−1.
Received: 8 August 1996/Revised: 4 March 1997 相似文献
12.
Calcium-induced fusion of liposomes was studied with a view to understand the role of membrane tension in this process. Lipid
mixing due to fusion was monitored by following fluorescence of rhodamine-phosphatidyl-ethanolamine incorporated into liposomal
membrane at a self-quenching concentration. The extent of lipid mixing was found to depend on the rate of calcium addition:
at slow rates it was significantly lower than when calcium was injected instantly. The vesicle inner volume was then made
accessible to external calcium by adding calcium ionophore A23187. No effect on fusion was observed at high rates of calcium
addition while at slow rates lipid mixing was eliminated. Fusion of labeled vesicles with a planar phospholipid membrane (BLM)
was studied using fluorescence microscopy. Above a threshold concentration specific for each ion, Ca2+, Mg2+, Cd2+ and La3+ induce fusion of both charged and neutral membranes. The threshold calcium concentration required for fusion was found to
be dependent on the vesicle charge, but not on the BLM charge. Pretreatment of vesicles with ionophore and calcium inhibited
vesicle fusion with BLM. This effect was reversible: chelation of calcium prior to the application of vesicle to BLM completely
restored their ability to fuse. These results support the hypothesis that tension in the outer monolayer of lipid vesicle
is a primary reason for membrane destabilization promoting membrane fusion. How this may be a common mechanism for both purely
lipidic and protein-mediated membrane fusion is discussed.
Received: 27 September 1999/Revised: 22 March 2000 相似文献
13.
One of the most important omissions in recent evolutionary theory concerns how eukaryotes could emerge and evolve. According
to the currently accepted views, the first eukaryotic cell possessed a nucleus, an endomembrane system, and a cytoskeleton
but had an inefficient prokaryotic-like metabolism. In contrast, one of the most ancient eukaryotes, the metamonada Giardia lamblia, was found to have formerly possessed mitochondria. In sharp contrast with the traditional views, this paper suggests, based
on the energetic aspect of genome organization, that the emergence of eukaryotes was promoted by the establishment of an efficient
energy-converting organelle, such as the mitochondrion. Mitochondria were acquired by the endosymbiosis of ancient α-purple
photosynthetic Gram-negative eubacteria that reorganized the prokaryotic metabolism of the archaebacterial-like ancestral
host cells. The presence of an ATP pool in the cytoplasm provided by this cell organelle allowed a major increase in genome
size. This evolutionary change, the remarkable increase both in genome size and complexity, explains the origin of the eukaryotic
cell itself.
The loss of cell wall and the appearance of multicellularity can also be explained by the acquisition of mitochondria. All
bacteria use chemiosmotic mechanisms to harness energy; therefore the periplasm bounded by the cell wall is an essential part
of prokaryotic cells. Following the establishment of mitochondria, the original plasma membrane-bound metabolism of prokaryotes,
as well as the funcion of the periplasm providing a compartment for the formation of different ion gradients, has been transferred
into the inner mitochondrial membrane and intermembrane space. After the loss of the essential function of periplasm, the
bacterial cell wall could also be lost, which enabled the naked cells to establish direct connections among themselves. The
relatively late emergence of mitochondria may be the reason why multicellularity evolved so slowly.
Received: 29 May 1997 / Accepted: 9 October 1997 相似文献
14.
The general purpose of this theoretical work is to contribute to understand the physiological role of the electrogenic properties
of the sodium pump, by studying a dynamic model that integrates diverse processes of ionic and water transport across the
plasma membrane. For this purpose, we employ a mathematical model that describes the rate of change of the intracellular concentrations
of Na+, K+ and Cl−, of the cell volume, and of the plasma membrane potential (V
m
). We consider the case of a nonexcitable, nonpolarized cell expressing the sodium pump; Na+, K+, Cl− and water channels, and cotransporters of KCl and NaCl in its plasma membrane. We particularly analyze here the conditions
under which the physiological V
m
can be generated in a predominantly electrogenic fashion, as a result of the activity of the sodium pump. A major conclusion
of this study is that, for the cell model considered, a low potassium permeability is not a sufficient condition for a predominantly
electrogenic generation of the V
m
by the sodium pump. The presence of an electroneutral exchange of Na+ and K+ represents a necessary additional requirement.
Received: 8 September 1999/Revised: 21 March 2000 相似文献
15.
The present study deals with photomodification of the electrical properties of the plasma membrane of an epithelial cell
line (opossum kidney (OK) cells). The effect of photofrin II (previously investigated) is compared with that of 5 other membrane-active
sensitizers: sulfonated Zn-phthalocyanine, merocyanine 540, rose bengal, methylene blue and protoporphyrin IX (an endogenous
sensitizer induced by addition of its biosynthetic precursor 5-aminolaevulinic acid). The study was performed in order to
investigate whether photomodification of the ion transport properties of the plasma membrane by membrane-active sensitizers
is a general and early event in cellular photosensitization. The changes in the electrical properties were monitored by application
of the whole-cell and the inside-out configuration of the patch-clamp technique.
Illumination in the presence of the compounds (apart from merocyanine 540) gave rise to similar changes of the electrical
properties of the membrane: depolarization of the membrane potential, inactivation of a large-conductance, Ca2+-dependent K+-channel (maxi-KCa), and a strong increase of the leak conductance of the membrane. This similarity indicates the general character of the functional
photomodifications by membrane-active sensitizers previously reported for photofrin II.
Received: 5 September 2000/Revised: 28 December 2000 相似文献
16.
Biodegradable pH-sensitive surfactants (BPS) are a unique family of easily metabolized compounds that demonstrate pH-dependent
surface activity. These agents, in combination with other delivery systems, have demonstrated effects in enhancing transnucleic
acid activity. The increased activity has been hypothesized to occur from a release of endosomal contents. Simply, the BPS
delivery system containing nucleic acids enters the cell through an endocytotoic process. It encounters an acidic pH and becomes
surface active leading to defects in the endosomal membrane. In the current study, an in vitro model membrane was used to
better understand the liposome defect mechanisms that BPS elicit. Using this system, it is shown that BPS can induce both
liposome fusion and rupture depending upon the pH and mole ratio of BPS to membrane lipids. Futhermore, liposome fusion induced
by BPS was dependent on the total numbers of liposome particles while rupture was independent of interacting liposome particles.
The generated data indicate that BPS agents act differently from other typical surface active agents and fuosgenic compounds.
Instead of facilitating membrane fusion through the hexagonal II phase, BPS appeared to contribute and participate in the
membrane fusion at different stages.
Received: 18 February 1998/Revised: 14 July 1998 相似文献
17.
The charge-pulse relaxation spectrum of nonperfused and perfused (turgescent) cells of the giant marine alga Ventricaria ventricosa showed two main exponential decays with time constants of approximately 0.1 msec and 10 msec, respectively, when the cells were bathed in artificial sea water (pH 8). Variation of the external pH did not change the relaxation pattern (in contrast to other giant marine algae). Addition of nystatin (a membrane-impermeable and pore-forming antibiotic) to the vacuolar perfusion solution resulted in the disappearance of the slow exponential, whereas external nystatin decreased dramatically the time constant of the fast one. This indicated (by analogy to corresponding experiments with Valonia utricularis, J. Wang, I. Spiess, C. Ryser, U. Zimmermann, J. Membrane Biol. 157: 311-321, 1997) that the fast relaxation must be assigned to the RC-properties of the plasmalemma and the slow one to those of the tonoplast. Consistent with this, external variation of [K+]o or of [Cl-]o as well as external addition of K+- or Cl--channel/carrier inhibitors (TEA, Ba2+, DIDS) affected only the fast relaxation, but not the slow one. In contrast, addition of these inhibitors to the vacuolar perfusion solution had no measurable effect on the charge-pulse relaxation spectrum. The analysis of the data in terms of the "two membrane model" showed that K+- and (to a smaller extent) Cl--conducting elements dominated the plasmalemma conductance. The analysis of the charge-pulse relaxation spectra also yielded the following area-specific data for the capacitance and the conductance for the plasmalemma and tonoplast (by assuming that both membranes have a planar surface): (plasmalemma) Cp = 0.82 * 10(-2) F m-2, Rp = 1.69 * 10(-2) Omega m2, Gp = 5.9 * 10(4) mS m-2, (tonoplast) Ct = 7. 1 * 10(-2) F m-2, Rt = 14.9 * 10(-2) Omega m2 and Gt = 0.67 * 10(4) mS m-2. The electrical data for the tonoplast show that (in contrast to the literature) the area-specific membrane resistance of the tonoplast of these marine giant algal cells is apparently very high as reported already for V. utricularis. The exceptionally high value of the area-specific capacitance could be explained - among other interpretations - by assuming a 9-fold enlargement of the tonoplast surface. The hypothesis of a multifolded tonoplast was supported by transmission electronmicroscopy of cells fixed under maintenance of turgor pressure and of the electrical parameters of the membranes. This finding indicates that the tonoplast of this species exhibited a sponge-like appearance. Taking this result into account, it can be easily shown that the tonoplast exhibits a high-resistance (1.1 Omega m2). Vacuolar membrane potential measurements (performed in parallel with charge-pulse relaxation studies) showed that the potential difference across the plasmalemma was mainly controlled by the external K+-concentration which suggested that the resting membrane potential of the plasmalemma is largely a K+-diffusion potential. After permeabilization of the tonoplast with nystatin the potential of the intact membrane barrier dropped from about slightly negative or positive (-5.1 to +18 mV, n = 13) to negative values (-15 up to -68 mV; n = 8). This indicated that the cytoplasm of V. ventricosa was apparently negatively charged relative to the external medium. Permeabilization of the plasmalemma by addition of external nystatin resulted generally in an increase in the potential to slightly more positive values (-0.8 to +4.3 mV; n = 5), indicating that the vacuole is positively charged relative to the cytoplasm. These findings apparently end the long-term debate about the electrical properties of V. ventricosa. The results presented here support the findings of Davis (Plant Physiol. 67: 825-831, 1981), but are contrary to the results of Lainson and Field (J. Membrane Biol. 29: 81-94, 1976). 相似文献
18.
The cell surface is a mechanobiological unit that encompasses the plasma membrane, its interacting proteins, and the complex underlying cytoskeleton. Recently, attention has been directed to the mechanics of the plasma membrane, and in particular membrane tension, which has been linked to diverse cellular processes such as cell migration and membrane trafficking. However, how tension across the plasma membrane is regulated and propagated is still not completely understood. Here, we review recent efforts to study the interplay between membrane tension and the cytoskeletal machinery and how they control cell form and function. We focus on factors that have been proposed to affect the propagation of membrane tension and as such could determine whether it can act as a global or local regulator of cell behavior. Finally, we discuss the limitations of the available tool kit as new approaches that reveal its dynamics in cells are needed to decipher how membrane tension regulates diverse cellular processes. 相似文献
19.
K-Cl cotransport (COT), a ouabain-insensitive, Cl-dependent bidirectional K flux, is ubiquitously present in all cells, plays
a major role in ion and volume homeostasis, and is activated by cell swelling and a variety of chemical interventions. Lithium
modulates several cation transport pathways and inhibits phospholipid turnover in red blood cells (RBCs). Lithium also inhibits
K-Cl COT by an unknown mechanism. To test the hypothesis whereby Li inhibits swelling-activated K-Cl COT by altering either
its osmotic response, its regulation, or by competing with K for binding sites, low K (LK) sheep (S) RBCs were loaded with
Li by Na/Li exchange or the cation ionophore nystatin. K-Cl COT was measured as the Cl-dependent, ouabain-insensitive K efflux
or Rb influx. The results show that Li altered the cell morphology, and increased both cell volume and diameter. Internal
(Li
i
) but not external (Li
o
) Li inhibited swelling-activated K-Cl COT by 85% with an apparent K
i
of ∼7 mm. In Cl, Li
i
decreased K efflux at relative cell volumes between 0.9 and 1.2, and at external pHs between 7.2 and 7.4. Li
i
reduced the V
max
and increased the K
m
for K efflux in Cl. Furthermore, Li
i
increased the production of diacylglycerol in a bimodal fashion, without significant effects on the phosphatidylinositol
concentration, and revealed the presence of a complete PI cycle in LK SRBCs. Finally, phorbol ester treatment and PD89059,
an inhibitor of mitogen-activated protein kinase (ERK2) kinase, caused a time-dependent inhibition of K-Cl COT. Hence, Li
i
appears to inhibit K-Cl COT by acting at an allosteric site on the transporter or its putative regulators, and by modulation
of the cellular phospholipid metabolism and a PKC-dependent regulatory pathway, causes an altered response of K-Cl COT to
pH and volume.
Received: 1 November 1999/Revised: 6 June 2000 相似文献
20.
Membrane trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is supposed to be an important mechanism
controlled by the intracellular messenger cAMP. This has been shown with fluorescence techniques, electron microscopy and
membrane capacitance measurements. In order to visualize protein insertion we applied atomic force microscopy (AFM) to inside-out
oriented plasma membrane patches of CFTR-expressing Xenopus laevis oocytes before and after cAMP-stimulation. In a first step, oocytes injected with CFTR-cRNA were voltage-clamped, verifying
successful CFTR expression. Water-injected oocytes served as controls. Then, plasma membrane patches were excised, placed
(inside out) on glass and scanned by AFM. Before cAMP-stimulation plasma membranes of both water-injected and CFTR-expressing
oocytes contained about 200 proteins per μm2. Molecular protein masses were estimated from molecular volumes measured by AFM. Before cAMP-stimulation, protein distribution
showed a peak value of 11 nm protein height corresponding to 475 kDa. During cAMP-stimulation with 1 mm isobutylmethylxanthine (IBMX) plasma membrane protein density increased in water-injected oocytes to 700 proteins per μm2 while the peak value shifted to 7 nm protein height corresponding to 95 kDa. In contrast, CFTR-expressing oocytes showed
after cAMP-stimulation about 400 proteins per μm2 while protein distribution exhibited two peak values, one peak at 10 nm protein height corresponding to 275 kDa and another
one at 14 nm corresponding to 750 kDa. They could represent heteromeric protein clusters associated with CFTR. In conclusion,
we visualized plasma membrane protein insertion upon cAMP-stimulation and quantified protein distribution with AFM at molecular
level. We propose that CFTR causes clustering of plasma membrane proteins.
Received: 11 September 2000/Revised: 13 December 2000 相似文献