Non-equilibration of hydrostatic pressure in blebbing cells

Abstract

Letter to NatureCurrent models for protrusive motility in animal cells focus on cytoskeleton-based mechanisms, where localized protrusion is driven by local regulation of actin biochemistry 1-3 . In plants and fungi, protrusion is driven primarily by hydrostatic pressure 4-6 . For hydrostatic pressure to drive localized protrusion in animal cells 7,8 , it would have to be locally regulated, but current models treating cytoplasm as an incompressible viscoelastic continuum 9 or viscous liquid 10 require that hydrostatic pressure equilibrates essentially instantaneously over the whole cell. Here, we use cell blebs as reporters of local pressure in the cytoplasm. When we locally perfuse blebbing cells with cortexrelaxing drugs to dissipate pressure on one side, blebbing continues on the untreated side, implying non-equilibration of pressure on scales of ~10μm and ~10sec. We can account for localization of pressure by considering the cytoplasm as a contractile, elastic network infiltrated by cytosol. Motion of the fluid relative to the network generates spatially heterogenous transients in the pressure field, and can be described in the framework of poroelasticity 11,12 .Blebs are large, approximately spherical deformations of the cell surface that form and disappear on a time-scale of tens of seconds. Though less studied than lamellipodial or filopodial protrusion, blebbing is a common phenomenon during apoptosis 13 , cytokinesis 14, 15 and cell movement 16,17 . Blebs are thought to be initiated by rupture of the plasma membrane from the underlying cytoskeleton (Supplementary figure S3), followed by inflation of the detached membrane by intracellular fluid flow 18,19 . Subsequent bleb retraction is driven by assembly and contraction of a cortex within the newly formed bleb. Blebbing requires nonuniform behaviour of the membrane and cortex, that must reflect either a globally uniform hydrostatic pressure combined with local nucleation of membrane detachment from the cytoskeleton, or non-uniform pressure leading to local rupture of membrane-cytoskeleton attachments in regions of high pressure. To distinguish these possibilities, we studied the effect of local disruption of cortical contraction or integrity induced by drug perfusion over part of a cell, in a filamin-depleted melanoma cell line (M2) that blebs extensively and continuously 20 .We first confirmed that the cell volume stays approximately constant during blebbing (Supplementary data, video S12 and figure S4) 18 , implying that blebbing is driven primarily by flow of fluid within the cell rather than water crossing the plasma membrane. We then confirmed that bleb inflation represents ballooning out of the plasma membrane when it detaches from the actin cortex by simultaneously imaging the cortex and the cell membrane (Supplementary figure S3). During bleb inflation, GFP-actin was not enriched at the bleb surface compared to its interior, implying lack of a cytoskeleton in the bleb ( Figure 1A, t=0,22s). As inflation slowed, a cortical cytoskeleton assembled ...

Implications of a poroelastic cytoplasm for the dynamics of animal cell shape

“…A simple but direct indication of how stresses and pressures may be out-of-equilibrium in cells over distances smaller than a cell length can be seen in an experiment where liquid was deliberately forced through cells into which passivated quantum dots had been microinjected using an external osmotic gradient (figure 4, GC, TM and LM, manuscript in preparation) using the local perfusion apparatus described in [29]). We found that two parts of the cytoplasm, separated by as little as ~5μm, could be strongly out of hydrodynamic equilibrium.…”

“…Blebs in most cells appear directionally uncontrolled, though blebbing of the leading edge thought to power directed motility in certain embryonic [37,38], amoeboid [39], and metastatic tumour cells [40][41][42][43]. Blebs, that protrude at rates of ~0.4 μm.s −1 [29], are more dramatic than standard, controlled protrusive events at the leading edge of motile cells. For example, lamellipodia protrude at ~0.15 μm.s −1 in rapidly moving fish keratocytes [44], and slower in most other cell types.…”

“…However, the complexity of ion homeostasis, multiple effects of changing ion concentrations in the cytoplasm, and a requirement for cytoskeleton to localize ion transporters and vice versa, will make it challenging to interpret such experiments. Local perfusion of inhibitors may be especially useful, since this will minimize systemic effects of changes in ion concentration and physical state in the bulk cytoplasm [29,64,65].…”

Finite-Element Stress Analysis of a Multicomponent Model of Sheared and Focally-Adhered Endothelial Cells

“…These models use average values of mechanical constants which arise from complex cellular architecture. Experimentally applied forces and readouts of deformation suggest that cell responses to stress can be modeled using linear elasticity, 8 tensegrity, 53 poroelasticity, 10 viscoelasticity, 30 power-law rheology, 32 and scale-free glassy rheology 3 depending on the magnitude and mode of force application. Finite element analysis remains the most effective tool to apply these constitutive frameworks to complex loading protocols and geometries; a process which is necessary in order to assess cellular responses to forces on multiple spatial and temporal scales.…”

Hydraulic fracturing in cells and tissues: fracking meets cell biology

“…An appealing mechanism is hydraulic fracturing caused by a transient contraction of the actomyosin cytoskeleton [4]. This mechanism is based on the idea that the cell interior is poroelastic; a local contraction of the cytoskeleton causes a local pressure buildup that slowly relaxes through water flow in the cytoskeletal network (Fig.…”