Neuronal Plasma Membrane Dynamics Evoked by Osmomechanical Perturbations

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     

Nuclear centering in Spirogyra: force integration by microfilaments along microtubules

The contribution of microtubules and microfilaments to the cytomechanics of transverse nuclear centering were investigated in the charophycean green alga Spirogyracrassa (Zygnematales). Cytoplasmic strands of enhanced rigidity and fasciate appearance radiate from the rim of the lenticular nucleus through the vacuole, frequently split once or twice and are attached to the helical chloroplast bands in the peripheral cytoplasm. The nucleus is encased in tubulin and a web of F-actin. Bundles of microtubules, emerging from the nuclear rim, are organized into dividing fascicles within the strands and reach to the inner surface of the chloroplast envelope. Organelles are translocated in both directions along similarly arranged fascicles of microfilament bundles which extend from the nucleus to the peripheral actin cytoskeleton. Application of microtubule- and/or microfilament-depolymerizing drugs affected the position of the nucleus only slowly, but in distinct ways. The differential effects suggest that nuclear centering depends on the tensional integrity of the perinuclear scaffold, with microfilaments conveying tension along stabilized microtubules and the actin cytoskeleton integrating the translocation forces generated within the scaffold. Received: 9 December 1996 / Accepted: 29 April 1997     

Establishment of a three-dimensional culture and mechanical loading system for skeletal myoblasts

Establishment of a three-dimensional (3-D) culture and mechanical loading system which simulates the in vivo environment is critical in cytomechanical studies. The present article attempts to do this by integrating porous PLGA scaffolds with a four-point bending strain unit. Three types of PLGA scaffolds with three average pore sizes were synthesized, i.e., type I (60-88 μm), type II (88-100 μm) and type III (100-125 μm). To establish the 3-D mechanical loading system, PLGA membrane was integrated with conventional force-loading plates and the third passage skeletal myoblasts from neonatal Sprague-Dawley (SD) rats were seeded. Small PLGA membranes were put in 24-well plates followed by cell implantation and MTT assay was performed on days 1, 2, 4, 6 and 8 to compare biocompatibility of the three types of scaffolds. After 3 days’ culture, many more cells had grown in type II than in type I or type III under fluorescence microscopy. In the MTT assay, OD of type II was significantly higher (P < 0.05) than the other two, especially at the early stage. As type II proved to be the best among the three, it was used as the scaffold in the preliminary mechanical loading study and 4000 μstrain cyclic uniaxial strain was imposed. The system worked well and it was found that short to median time of stretching enhances while prolonged time of stretching inhibits cell proliferative activity of the 3-D cultured skeletal myoblasts(P < 0.05). It is concluded that the combination of PLGA scaffolds with a four-point bending strain unit provides a satisfactory 3-D mechanical loading system.     

Gravisusception by buoyancy: a mechanism ubiquitous among fungi?

Gravitropism is ubiquitous among the fungal taxa; however, the mechanism(s) of gravisusception have overall remained obscure so far. In the vegetative sporangiophore of the zygomycete Phycomyces blakesleeanus some 200 large lipid globules form a conspicuous spherical complex which is positioned in a dense mesh of filamentous actin about 100 microm below the growing tip of the apex. Experimental suppression of that complex by transient growth at low temperature greatly diminishes the gravitropic response of the sporangiophore. With respect to size and abundance of the globules, the complex of lipid globules meets basic physical criteria for a possible function of gravisusception. Accumulations of similar lipid globules of critical size are documented in the apex of gravitropically growing hyphae of the endomycorrhizal fungus Gigaspora margarita (Glomeromycota) and have been described in the hyphal apices of members of various fungal phyla. We suppose that--in contrast to plants which use starch as a carbon storage and amyloplasts as statoliths--the fungi utilise the buoyancy of carbon-storing oil droplets for gravisusception.     

Cell Surface Area Regulation and Membrane Tension

The beautifully orchestrated regulation of cell shape and volume are central themes in cell biology and physiology. Though it is less well recognized, cell surface area regulation also constitutes a distinct task for cells. Maintaining an appropriate surface area is no automatic side effect of volume regulation or shape change. The issue of surface area regulation (SAR) would be moot if all cells resembled mammalian erythrocytes in being constrained to change shape and volume using existing surface membrane. But these enucleate cells are anomalies, possessing no endomembrane. Most cells use endomembrane to continually rework their plasma membrane, even while maintaining a given size or shape. This membrane traffic is intensively studied, generally with the emphasis on targeting and turnover of proteins and delivery of vesicle contents. But surface area (SA) homeostasis, including the controlled increase or decrease of SA, is another of the outcomes of trafficking. Our principal aims, then, are to highlight SAR as a discrete cellular task and to survey evidence for the idea that membrane tension is central to the task. Cells cannot directly ``measure' their volume or SA, yet must regulate both. We posit that a homeostatic relationship exists between plasma membrane tension and plasma membrane area, which implies that cells detect and respond to deviations around a membrane tension set point. Maintenance of membrane strength during membrane turnover, a seldom-addressed aspect of SA dynamics, we examine in the context of SAR. SAR occurs in both animal and plant cells. The review shows the latter to be a continuing source of groundbreaking work on tension-sensitive SAR, but is principally slanted to animal cells. Received: 1 May 2000/Revised: 14 August 2000     

The local cytomechanical properties of growing pollen tubes correspond to the axial distribution of structural cellular elements

Morphological studies of pollen tubes have shown that the configuration of structural cellular elements differs between the growing apex and the distal part of the cell. This polarized cellular organization reflects the highly anisotropic growth behavior of this tip growing cell. Accordingly, it has frequently been postulated that physical properties of pollen tubes such as cell wall plasticity should show anisotropic distribution, but no experimental evidence for this has been published hitherto. Using micro-indentation techniques, we quantify pollen tube resistance to lateral deformation forces and analyze its visco-elasticity as a function of distance from the growing apex. Our studies reveal that cellular stiffness is significantly higher at the distal portion of the cell. This part of the cell is also completely elastic, whereas the apex shows a visco-elastic component upon deformation. To relate these data to the architecture of the particular pollen tube investigated in this study, Papaver rhoeas, we analyzed the distribution of cell wall components such as pectin, callose, and cellulose as well as the actin cytoskeleton in this cell using fluorescence label. Our data revealed that, in particular, the degree of pectin methyl esterification and the configuration of the actin cytoskeleton correlate well with the distribution of the physical properties on the longitudinal axis of the cell. This suggests a role for these cellular components in the determination of the cytomechanics of pollen tubes.     

Pectin and the role of the physical properties of the cell wall in pollen tube growth of<Emphasis Type="Italic"> Solanum chacoense</Emphasis>

The cell wall is one of the structural key players regulating pollen tube growth, since plant cell expansion depends on an interplay between intracellular driving forces and the controlled yielding of the cell wall. Pectin is the main cell wall component at the growing pollen tube apex. We therefore assessed its role in pollen tube growth and cytomechanics using the enzymes pectinase and pectin methyl esterase (PME). Pectinase activity was able to stimulate pollen germination and tube growth at moderate concentrations whereas higher concentrations caused apical swelling or bursting in Solanum chacoense Bitt. pollen tubes. This is consistent with a modification of the physical properties of the cell wall affecting its extensibility and thus the growth rate, as well as its capacity to withstand turgor. To prove that the enzyme-induced effects were due to the altered cell wall mechanics, we subjected pollen tubes to micro-indentation experiments. We observed that cellular stiffness was reduced and visco-elasticity increased in the presence of pectinase. These are the first mechanical data that confirm the influence of the amount of pectins in the pollen tube cell wall on the physical parameters characterizing overall cellular architecture. Cytomechanical data were also obtained to analyze the role of the degree of pectin methyl-esterification, which is known to exhibit a gradient along the pollen tube axis. This feature has frequently been suggested to result in a gradient of the physical properties characterizing the cell wall and our data provide, for the first time, mechanical support for this concept. The gradient in cell wall composition from apical esterified to distal de-esterified pectins seems to be correlated with an increase in the degree of cell wall rigidity and a decrease of visco-elasticity. Our mechanical approach provides new insights concerning the mechanics of pollen tube growth and the architecture of living plant cells.     

In the beginning there were soft collagen-cell gels: towards better 3D connective tissue models?

In the 40 years since Elsdale and Bard's analysis of fibroblast culture in collagen gels we have moved far beyond the concept that such 3D fibril network systems are better models than monolayer cultures. This review analyses key aspects of that progression of models, against a background of what exactly each model system tries to mimic. This story tracks our increasing understanding of fibroblast responses to soft collagen gels, in particularly their cytoskeletal contraction, migration and integrin attachment. The focus on fibroblast mechano-function has generated models designed to directly measure the overall force generated by fibroblast populations, their reaction to external loads and the role of the matrix structure. Key steps along this evolution of 3D collagen models have been designed to mimic normal skin, wound repair, tissue morphogenesis and remodelling, growth and contracture during scarring/fibrosis. As new models are developed to understand cell-mechanical function in connective tissues the collagen material has become progressively more important, now being engineered to mimic more complex aspects of native extracellular matrix structure. These have included collagen fibril density, alignment and hierarchical structure, controlling material stiffness and anisotropy. But of these, tissue-like collagen density is key in that it contributes to control of the others. It is concluded that across this 40 year window major progress has been made towards establishing a family of 3D experimental collagen tissue-models, suitable to investigate normal and pathological fibroblast mechano-functions.     

Osmotic dilution stimulates axonal outgrowth by making axons more sensitive to tension

Mechanical tension is a potent stimulator of axonal growth rate, which is also stimulated by osmotic dilution. We wished to determine the relationship, if any, between osmotic stimulation and tensile regulation of axonal growth. We used calibrated glass needles to apply constant force to elongate axons of cultured chick sensory neurons. We find that a neurite being pulled at a constant force will grow 50–300% faster following a 50% dilution of inorganic ions in the culture medium. That is, osmotic dilution appears to cause axons to increase their sensitivity to applied tensions. Experimental interventions suggest that this effect is not mediated by dilution of extracellular calcium, or to osmotic stimulation of adenylate cyclase, or to osmotic stimulation of mechanosensitive ion channels. Rather, experiments measuring the static tension normally borne by neurites suggest a direct mechanical effect on the cytoskeletal proteins of the neurite shaft. Our results are consistent with a formal thermodynamic model for axonal growth in which removing a compressive load on axonal microtubules promotes their assembly, thus promoting axonal elongation.