© 2001 by The Society for Integrative and Comparative Biology
Mechanosensitive Membrane Traffic and an Optimal Strategy for Volume and Surface Area Regulation in CNS Neurons1
1 Neurosciences, Ottawa Health Research Institute Ottawa Hospital, 725 Parkdale Ave., Ottawa, Ontario K1Y 4K9, Canada
 |
SYNOPSIS |
|---|
TOP
 SYNOPSIS BACKGROUNDVOLUME AND SURFACE AREA...QUESTIONS FOR TESTING THE...VSAR-CNS IN RELATION TO...References |
|---|
Extrapolating from a body of work on isolated neurons, a model is suggested for how central nervous system (CNS) neurons in situ may handle local swelling and shrinking perturbations so as to interfere minimally with the primary role of neurons, synaptic information processing. The strategy used for osmoregulatory membrane adjustments may first come into play during ontogeny, when it could provide membrane tension sensitive membrane trafficking in arborized neurons subjected to morphogenetic forces.
|
BACKGROUND |
|---|
|
TOP
SYNOPSISBACKGROUNDVOLUME AND SURFACE AREA...QUESTIONS FOR TESTING THE...VSAR-CNS IN RELATION TO...References |
|---|
Neuronal architecture—the carefully established arborizations of neurons, along with the minutiae of their neuron-neuron contacts—is key to information flow and information storage in the CNS. Though CNS neurons are protected from osmotic extremes, bouts of intense neural activity produce local swelling and shrinking influences in the form of extracellular [K+] and intracellular osmolyte changes. Given the massively reticular architecture of the CNS, it is unsurprising that monitoring cell volume and surface area dynamics in the CNS is dauntingly difficult. Compounding the difficulty, however, has been an unsettling disjunction between phenomena observed in volume-perturbed CNS cells isolated in culture and phenomena evident from CNS cells monitored collectively (i.e., in brain slice). Regulatory volume decrease—the set of cell-mediated processes by which swollen cells reshrink towards their normal volume in the continued presence of hyposmotic medium—is reported for neurons (Morán et al., 1997
), glia (Morán et al., 1996) and endothelial cells (Hara et al., 1999) in culture, but to date, using a variety of methods, comparable regulatory volume decrease has not been detected in brain slices (e.g., Andrew et al., 1997, 1999). Is it that brain cells, though possessed of machinery for volume regulation, opt, when in situ, not to use it? Or is it that, in situ, neurons and their neighbors finesse their volume changes in such a way that regulatory events get overlooked? This possibility—that CNS cell volume regulation is cryptic rather than absent—is considered below.
An obvious and important difference between culture and in situ conditions is that in situ, CNS cells adhere to other cells over their entire surface, mostly via an intervening extracellular matrix of variable thickness. As Van Essen (1997) points out, the simple fact that synaptosomes survive mechanical homogenization of CNS tissue attests to the adhesive strength of extracellular matrix in synaptic zones. In culture, when adhesion is prevented, neurons retrieve their processes and round up; evidently, even on abiotic substrata, adhesion forces excede the steady-state counterforces generated by cytoskeletal tension and compression. Cultured cells are, however, non-adherent over most of their surface. Cells in suspension are completely non-adherent. Cell volume studies frequently use both but the non-adherent membrane expanses of theses cells may exhibit qualitatively (and quantitatively) abnormal responses to osmomechanical perturbations. Many cell functions depend pivotally on signals transduced at adhesive contacts where, via sketchily-understood mechanisms, mechanical forces control cellular "decision making" (Condron and Zinn, 1997; Chicurel et al., 1998; Huang and Ingber, 1999). In situ, the entire surface of neurons and glia is adherent surface. When cultured cells are subjected to osmotic challenges, therefore, particular note should be paid to how their adherent surface membrane responds to the changing mechanical forces.
One might well expect the adherent surface to be the most stable place in a cultured neuron, and so it appears during swelling, but when cultured neurons then reshrink, their adherent surface is anything but quiescent. First, one makes this startling observation: the adherent surface vacuolates! This is not a "sick cell" artefact, but a highly reproducible and, in healthy cells, transient, phenomenon. But what appear to be "vacuoles" are in fact invaginations of adherent surface plasma membrane, somehow drawn inward by forces from the shrinking cytoplasm. Accordingly, they are termed "vacuole-like dilations" (VLDs), not vacuoles. Similar invaginations develop in renal cells during a 5 min cell-mediated shrinkage (i.e., regulatory volume decrease) (Czekay et al., 1993; see Morris and Homann, 2001) as well as during slow cell-mediated volume reduction in neurons (Herring et al., 1999). Part of what makes VLDs transient is a second set of events also initiated at the adherent surface—the rapid re-arrangement of F-actin into circles of motile leading edge at the mouths of VLDs. The circle advances centripetally (Herring et al., 1999), in some instances internalizing the still-dilated VLD membrane, in others, causing it to shrink and disappear. Though counterintuitive when mistaken for vacuoles, VLDs seen correctly as transient invaginations of excess neuronal membrane (see Dai et al., 1998 for an example in hippocampal neurons) suggest a means whereby CNS cells in situ might cope with volume changes. These experiments and related work on membrane tension are reviewed in some detail in another review (Morris and Homann, 2001). Here I focus on a strategy VLDs have suggested for synapse-sparing volume and surface area regulation of neurons in the CNS ("VSAR-CNS"). This VSAR-CNS model is extrapolated from the 2-D situation of cultured neurons to the 3-D situation of CNS tissue. Interpolation is also required; having subjected neurons to non-physiological (large, abrupt) osmomechanical perturbations, we interpolate to the milder perturbations experienced within the CNS. Big step perturbations are justifiable if they can tease out underlying mechanisms (e.g., as with voltage clamp) and speculating via a model is warranted if it provides a framework for further experiments.
|
VOLUME AND SURFACE AREA REGULATION OF NEURONS IN THE CNS: A "VSAR-CNS" MODEL |
|---|
|
TOP
SYNOPSISBACKGROUNDVOLUME AND SURFACE AREA...QUESTIONS FOR TESTING THE...VSAR-CNS IN RELATION TO...References |
|---|
The VSAR-CNS model suggests how volume and surface area homeostasis could be achieved while minimally jeopardizing the raison d'être of central neurons, namely to be reliable conduits and repositories of information. The model assumes that neither established cell shapes nor established cell-cell contacts should be sacrificed in achieving neuronal VSAR. Additionally, it assumes that VSAR should be achieved without disrupting Ca-dependent synaptic events.
The model embodied in Figure 1 invokes no calcium signals, and indeed, the primary events of neuronal surface area and volume regulation are known to be calcium-independent. Capacitance measurements show that swelling-then-reshrinking neurons increase-then-decrease their surface area without need of calcium influx (Wan et al., 1995). Likewise, the swell/shrink-induced formation of VLDs is independent of intracellular and extracellular calcium levels (Herring et al., 1998). Instead, the membrane area changes and VLD formation are driven by mechanical forces, and in particular, it appears, by membrane tension. Calcium-independent, tension-driven surface area regulation is probably ancient as it is also evident in swelling and shrinking plant cells (Zorec and Tester, 1993; Homann, 1998; Homann and Thiel, 1999; Kubitscheck et al., 2000). What of volume regulation? Regulatory volume decrease in some, but not all, cell types is calcium-dependent; Fura-2 experiments indicate that mammalian CNS neurons are among those cells for which regulatory volume decrease requires no calcium signalling (Morán et al., 1997). Thus extraneous calcium signals that might compromise synaptic transmission and synaptic plasticity (learning, memory) can be dispensed with. As an aside, assuming that VSAR-CNS proceeds without calcium signals does not imply that a basal or "housekeeping" level of intracellular calcium is unnecessary. For example, VLD recovery (the F-actin dependent disappearance of VLDs alluded to above) is impaired when neurons become calcium-depleted (Reuzeau et al., 1995) and intracellular calcium below 50 nM renders neurons osmotically fragile (Herring et al., 1998; Dai et al., 1998).
|
An important feature of the model for volume and surface area regulation of neurons in the CNS (i.e., for VSAR-CNS) is that the process does not mechanically perturb (stretch or relocate) the 3-D co-ordinates of synapses. An electrically hyperactive neuron (or a cluster of such neurons) would swell and then reshrink (experiencing elevated and decreased membrane tension, respectively (Dai et al., 1998
) but the model predicts that actual swell/shrink movements of the cell would be confined to regions distinct from synaptic active zones. First, consider swelling in situ: small perineuronal spaces containing readily compressible extracellular matrix accommodate localized swelling protrusions, as depicted (Fig. 1a). Displaced extracellular fluid is handled by nearby capillaries. Importantly, a protrusion as just described involves extra-synaptic, not synaptic, plasma membrane. Localized protrusion is fostered because the relevant locations are pre-stocked with mechanically-accessible membrane reserves. As isolated neurons swell (Wan et al., 1995), they can produce distinct protrusions or cytoplasmic blebs (as opposed to membrane blebs) perhaps using such localized mechanically-accessible membrane reserves. A neuron may have several types of membrane. Putative "hot spots" of subsurface cisternae have been seen by electron microscopy (Fejtl et al., 1995). Subsurface membrane discs reported from in situ neurons (Cheng and Reese, 1987) may be recruited by elevated membrane tension. It may be germane that work with elongating plant protoplasts is suggesting that the secretory vesicles used for carrying cell wall material and the mechanosensitive (i.e., mechanically-accessible) exocytotic vesicles used for increasing membrane area constitute distinct populations (Thiel et al., 2000). Newly-invaginated VLD membrane is a source of membrane in reswelling cells (Reuzeau et al., 1995; Mills and Morris, 1998) and it is thought that spectrin membrane skeleton associated with the VLDs (Herring et al., 2000) may persist and allow them to exhibit "memory" (Reuzeau et al., 1995; Mills and Morris, 1998). This memory is seen with repeated swell/shrink cycles, since VLDs form repeatedly at the same discrete locations. In other words, for isolated neurons, SAR induced by osmotic perturbations occurs at discrete sites. This constitutes the basis for the discrete-sites feature of the model—and here the discrete sites are specifically extra-synaptic.
Another outcome of discrete site invagination in shrinking neurons is that a neuron can have a more-or-less static perimeter even while its cytoplasmic volume decreases (Fig. 1b). The consequence is that synaptic membrane would stay put, as depicted in the VSAR-CNS cartoon. Mechanical forces within the shrinking cytoplasm somehow foster retrieval (invagination) of plasma membrane at several points of low cell-cell adhesion. Initially, VLDs are drawn in as 
1 µm diameter tubes (Mills and Morris, 1998; Herring et al., 1999) and as shrinkage continues, they inflate, as if water exiting the cytoplasm flows preferentially across invaginating (flowing) membrane. Whatever the exact process, hydrostatic pressure in the VLD lumen is temporarily sufficient to dilate the invagination. In the process, excess surface area acquired in the preceding bout of swelling becomes VLD membrane. Figure 1b compares simple shrinkage (arrow with the X) with VLD-based shrinkage (arrow with the check). Deploying invaginated (excess) surface membrane to create a volume of extracellular space at a pseudo-intracellular location allows the neuron's cytoplasmic volume to decrease without retraction of its cell perimeter. Shrinkage in this mode is, thus, cryptic. The resulting avoidance of mechanical stress at synapses could be important for avoiding unwanted mechanically-induced fusion of docked transmitter-filled vesicles, since extreme shrinking stimuli can evidently elicit transmitter release (Rosenmund and Stevens, 1996; Chen and Grinell, 1997). Ideally, in VSAR-CNS both the general architecture of a neurons and the location of its synaptic active zones is unaffected by osmotic perturbations. There is a requirement for movement in the plane of the bilayer; diffusible bilayer molecules flow from low to high tension membrane regions (Mills and Morris, 1998). During shrinkage, flow will be towards the high tension invaginating VLD membrane, but this drift should have little impact on synaptic active zones, whose constituents are well anchored (e.g., Sheng and Pak, 1999) and may be in special lipid rafts (Bruses et al., 2001).
|
QUESTIONS FOR TESTING THE VSAR-CNS MODEL |
|---|
|
TOP
SYNOPSISBACKGROUNDVOLUME AND SURFACE AREA...QUESTIONS FOR TESTING THE...VSAR-CNS IN RELATION TO...References |
|---|
(1) Are there discrete extracellular spaces adjacent to neurons that would allow for small swelling protrusions, as proposed?
(2) Are extracellular matrix pockets soft enough to compress under the influence of a swelling protrusion (in other words, are extracellular lacunaea softer than adjacent adherent cells)?
(3) Adjacent to the lacunaea, are there membrane reserves available to expand the membrane area of a swelling protrusion?
(4) Using various vital marker dye regimes on brain slices, can one detect transient pseudo-intracellular dilations forming in shrinking neurons? In particular, do transient VLDs appear under conditions where one would expect shrinkage due to regulatory volume decrease?
(5) Do inhibitors of F-actin applied in situ lead to accumulation of VLDs after osmotic perturbations?
(6) In an in situ swelling or shrinking neuron respectively, do vital stain markers of synaptic zones all mutually move away from or towards each other, or do they, as predicted, remain in place?
|
VSAR-CNS IN RELATION TO NEURAL DEVELOPMENT AND NEURAL DYSFUNCTION |
|---|
|
TOP
SYNOPSISBACKGROUNDVOLUME AND SURFACE AREA...QUESTIONS FOR TESTING THE...VSAR-CNS IN RELATION TO...References |
|---|
Mechanosensitive membrane insertion and retrieval is crucial for VSAR-CNS. As Figure 2 implies, mechanosensitive membrane traffic could be a fine solution to problems first met by neurons during morphogenesis and enlargement of the CNS (Morris et al., 1997
). A single neuron will simultaneously be subjected to different tensions (above normal, normal, below normal) along different vectors. Simultaneously, along those neurites experiencing high tension, membrane would be inserted at elevated rates while, in retracting neurites of the same neuron, endocytosis would proceed at the high rates facilitated by low membrane tension (Fink and Cooper, 1996; Dai et al., 1997; Raucher and Sheetz, 1999). As suggested above for osmoregulatory adjustments, local membrane tension would act both as sensor and effector for growth-related SAR, without recourse to special chemical signalling.
|
The membrane skeleton proteins, spectrin and F-actin, form a continuous lining for invaginated VLD membrane. Spectrin is thought to provide an extensible passive mechanical support during invagination (Herring et al., 2000
), whereas F-actin, which is contractile by virtue of its interaction with myosin, allows the invaginated membrane to be actively reprocessed, returning the cell to its normal form (Reuzeau et al., 1995; Herring et al., 1999). Neurons rendered transiently hyperactive in situ (a side effect of therapeutic drugs) vacuolate transiently (Auer and Coulter, 1994) in a manner that suggests impaired VSAR is component of non-lethal excitotocity. During stroke, excitotoxic (Ca-mediated) damage to spectrin and F-actin is an early indicator of seriously compromised tissue. Neurons that survive a stroke and make effective repairs will need to avail themselves of VSAR, and VSAR demands a well-behaved membrane skeleton. For reasons unrelated to VSAR, serious consideration is being given to stroke interventions which target membrane skeleton proteins (Yokata et al., 1999; Endres et al., 1999). Given how little is understood about membrane skeleton and its dynamics in neurons or other CNS cells, the prospect of such therapies creates an urgent need to pay special attention to membrane skeleton in future studies of VSAR in the CNS.
|
ACKNOWLEDGMENTS |
|---|
Supported by a grant from the Heart and Stroke Foundation of Ontario (NA4436).
|
FOOTNOTES |
|---|
1 From the Symposium Osmoregulation: An Integrated Approach presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2000, at Atlanta, Georgia.
|
References |
|---|
|
TOP
SYNOPSISBACKGROUNDVOLUME AND SURFACE AREA...QUESTIONS FOR TESTING THE...VSAR-CNS IN RELATION TO...References |
|---|
Andrew, R. D., M. E. Lobinowich, and E. P. Osehobo. 1997. Evidence against volume regulation by cortical brain cells during acute osmotic stress. Exp. Neurol, 143:300-312.[CrossRef][Web of Science][Medline]
Andrew, R. D., C. R. Jarvis, and A. S. Obeidat. 1999. Potential sources of intrinsic optical signals imaged in live brain slices. Methods, 18:185-196.[CrossRef][Web of Science][Medline]
Auer, R. N., and K. C. Coulter. 1994. The nature and time course of neuronal vacuolation induced by the N-methyl-D-aspartate antagonist MK-801. Acta Neuropathol. (Berl.), 87:1-7.[Medline]
Bruses, J., N. Chauvet, and U. Rutishauser. 2001. Membrane lipid rafts are necessary for the maintenance of the (
)7 nicotinic acetylcholine receptor in somatic spines of ciliary neurons. J. Neurosci, 21:504-512.
Chen, B. M., and A. D. Grinnell. 1997. Kinetics, Ca2+ dependence, and biophysical properties of integrin-mediated mechanical modulation of transmitter release from frog motor nerve terminals. J. Neurosci, 17:904-916.
Cheng, T. P. O., and T. S. Reese. 1987. Recycling of plasmalemma in chick tectal growth cones. J. Neurosci, 7:1752-1759.[Abstract]
Chicurel, M. E., C. S. Chen, and D. E. Ingber. 1998. Cellular control lies in the balance of forces. Curr. Opin. Cell Biol, 10:232-239.[CrossRef][Web of Science][Medline]
Condron, B. G., and K. Zinn. 1997. Regulated neurite tension as a mechanism for determination of neuronal arbor geometries in vivo. Curr. Biol, 7:813-816.[CrossRef][Web of Science][Medline]
Czekay, R., E. Kinne-Saffran, and R. K. H. Kinne. 1993. Membrane traffic and sorbitol release during osmo- and volume regulation in isolated rat renal inner medullary collecting duct cells. Eur. J. Cell Biol, 63:20-31.
Dai, J., M. P. Sheetz, X. Wan, and C. E. Morris. 1998. Membrane tension in swelling and shrinking molluscan neurons. J. Neurosci, 18:6681-6692.
Dai, J., H. P. Ting-Beall, and M. P. Sheetz. 1997. The secretion-coupled endocytosis correlates with membrane tension changes in RBL 2H3 cells. J. Gen. Physiol, 110:1-10.
Endres, M., K. Fink., J. Zhu, N. E. Stagliano, V. Bondada, J. W. Geddes, T. Azuma, M. P. Mattson, D. J. Kwiatkowski, and M. A. Moskowitz. 1999. Neuroprotective effects of gelsolin during murine stroke. J. Clin. Invest, 103:347-354.[Web of Science][Medline]
Fejtl, M., D. H. Szarowski, D. Decker, K. Buttle, D. O. Carpenter, and J. N. Turner. 1995. Three-dimensional imaging and electrophysiology of live Aplysia neurons during volume perturbation: Confocal light and high-voltage electron microscopy. J. Microscop. Soc. Am, 1:75-85.
Fink, R. D., and M. S. Cooper. 1996. Apical membrane turnover is accelerated near cell-cell contacts in an embryonic epithelium. Dev. Biol, 174:180-189.[CrossRef][Web of Science][Medline]
Hara, E., P. S. Reinach, Q. Wen, P. Iserovich, and J. Fischbarg. 1999. Fluoxetine inhibits K(+) transport pathways (K(+) efflux, Na(+)–K(+)– 2Cl(–) cotransport, and Na(+) pump) underlying volume regulation in corneal endothelial cells. J. Membr. Biol, 171:75-85.[CrossRef][Web of Science][Medline]
Herring, T. L., I. M. Slotin, J. M. Baltz, and C. E. Morris. 1998. Neuronal swelling and surface area regulation: Elevated intracellular calcium is not a requirement. Am. J. Physiol, 274:C272-C281.
Herring, T. L., C. S. Cohan, E. A. Welnhofer, L. R. Mills, and C. E. Morris. 1999. F-actin at newly invaginated membrane in neurons: Implications for surface area regulation. J. Membr. Biol, 171:151-169.[CrossRef][Web of Science][Medline]
Herring, T. L., P. F. Juranka, J. McNally, H. Lesiuk, and C. E. Morris. 2000. The spectrin skeleton of newly-invaginated plasma membrane. J. Mus. Res. Cell. Mot, 21:67-77.[CrossRef][Web of Science][Medline]
Homann, U. 1998. Fusion and fission of plasma-membrane material accommodates for osmotically induced changes in the surface area of guard-cell protoplasts. Planta, 206:329-333.[CrossRef][Web of Science]
Homann, U., and G. Thiel. 1999. Unitary exocytotic and endocytotic events in guard-cell protoplasts during osmotic-driven volume changes. FEBS Lett, 460:495-499.[CrossRef][Web of Science][Medline]
Huang, S., and D. E. Ingber. 1999. The structural and mechanical complexity of cell-growth control. Nat. Cell Biol, 1: E131-E138.
Kubitscheck, U., U. Homann, and G. Thiel. 2000. Osmotic evoked shrinking of guard cell protoplasts causes retrieval of plasma membrane into the cytoplasm. Planta, 210:423-431.[CrossRef][Web of Science][Medline]
Mills, L. R., and C. E. Morris. 1998. Neuronal plasma membrane dynamics evoked by osmomechanical perturbations. J. Membr. Biol, 166:223-238.[CrossRef][Web of Science][Medline]
Morán, J., M. Sabanero, I. Meza, and H. Pasantes-Morales. 1996. Changes of actin cytoskeleton during swelling and regulatory volume decrease in cultured astrocytes. Am. J. Physiol, 271:C1901-C1907.
Morán, J., S. Morales-Mulia, A. Hernandez-Cruz, and H. Pasantes-Morales. 1997. Regulatory volume decrease and associated osmolyte fluxes in cerebellar granule neurons are calcium independent. J. Neurosci. Res, 47:144-154.[CrossRef][Web of Science][Medline]
Morris, C. E., and U. Homann. 2001. Cell surface area regulation and membrane tension. J. Membr. Biol, 179:79-102.[Web of Science][Medline]
Morris, C. E., H. Lesiuk, and L. R. Mills. 1997. How do neurons monitor their mechanical status? Biol. Bull, 192:118-120.
Raucher, D., and M. P. Sheetz. 1999. Membrane expansion increases endocytosis rate during mitosis. J. Cell Biol, 144:497-506.
Reuzeau, C., L. R. Mills, J. A. Harris, and C. E. Morris. 1995. Discrete and reversible vacuole-like dilations induced by osmomechanical perturbation of neurons. J. Membr. Biol, 145:33-47.[Web of Science][Medline]
Rosenmund, C., and C. F. Stevens. 1996. Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron, 16:1197-1207.[CrossRef][Web of Science][Medline]
Sheng, M., and D. T. Pak. 1999. Glutamate receptor anchoring proteins and the molecular organization of excitatory synapses. Ann. N. Y. Acad. Sci, 868:483-493.[CrossRef][Web of Science][Medline]
Thiel, G., J.-U. Sutter, and U. Homann. 2000. Ca2+-sensitive and Ca2+-insensitive exocytosis in maize coleoptile protoplasts. Pflügers Arch, 439: R152-R153.
Van Essen, D. C. 1997. A tension-based theory of morphogenesis and compact wiring in the central nervous system. Nature, 385:313-318.[CrossRef][Medline]
Wan, X., J. A. Harris, and C. E. Morris. 1995. Responses of neurons to extreme osmomechanical stress. J. Membr. Biol, 145:21-31.[Web of Science][Medline]
Yokota, M., E. Tani, S. Tsubuki, I. Yamaura, I. Nakagaki, S. Hori, and T. C. Saido. 1999. Calpain inhibitor entrapped in liposome rescues ischemic neuronal damage. Brain Res, 819:8-14.[CrossRef][Medline]
Zorec, R., and M. Tester. 1993. Rapid pressure driven exocytosis-endocytosis cycle in a single plant cell. FEBS Lett, 333:283-286.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||