Cytoskeleton Dynamics

How do blebs expand?

Expansion lasts between 5 to 30 seconds, following bleb initiation and preceding reformation of the cortical actomyosin cytoskeleton just beneath the membrane [1]. During this time, the bleb proceeds to grow as a result of actomyosin driven pressure, resulting in the influx of cytoplasm into the bleb. Cytoplasmic flow within the cell follows a fountain-like pattern through the inner and outer regions [2].

The cytoplasmic volume of the bleb increases in concert with its membrane surface area [3]. Expansion of the membrane surface area is enabled by lipid flow through the neck of the bleb [4][5]. If the growth of the bleb is too rapid to be facilitated by lipid flow alone, the membrane-cortex connection adjacent to the neck of the bleb can get further disrupted due to increased membrane tension [6].

Growing blebs are generally devoid of cortical actin, both in non-motile [7][8][9] and motile cells [10] [11] [12]. Instead, they possess a spectrin-based cytoskeleton, such as that observed in red blood cells [13]. The maximal size of the bleb is determined by its initial growth rate, membrane tension and the time required for repolymerization of the cortical cytoskeleton [7].

Motile Blebbing Expansion

Motile blebbing (as opposed to non-motile) occurs primarily at the leading edge. The stimulus and subsequent downstream signaling that initiates this polarization is not yet clear. However, two polarization models have been proposed, one based on the local membrane detachment and the other based on the local cortex rupture scenario [1]:

Local membrane detachment
In this case, polarization may result from discrete, local weakening of membrane-cortex attachments. This may may be the consequence of uneven distribution of the actin-membrane linker ERM (ezrin, radixin and moesin) to the rear of the cell. This model is supported by fixed Walker carcinosarcoma cells [14].

Local cortex rupture
In this case, preferential tearing at the leading edge may result from higher contractility at the rupture point [8]f, without the need for asymmetry in membrane-cortex attachments.

In both models, old cortex is disassembled at leading edge and new cortex is formed under the growing bleb. Since the leading edge cortex is always younger and more fragile, once the symmetry is broken, polarization continues.

Adherence

In motile blebbing, cells move by exerting a force against the underlying substrate. In lamellipodial motility, this is achieved through adherence of lamellipodia to the substrate. However, in blebbing motility, the mechanism is still unknown. Two models have been proposed [1]:

Weak substrate adhesion
The blebbing cell creates weak adhesions to the substrate. This model is supported by findings from Walker carcinosarcoma cells, showing loose cell-substrate contacts during motile blebbing [15]. Loose cell-cell contacts have also been observed, with contacts spanning a greater distance than that of tight or adherens junctions [16].

Perpendicular force generation
If a motile, blebbing cell is in a confined environment, such as between two glass cover slips, it is able to migrate without the use of cell-substrate adhesions. This is due to the generation of forces perpendicular to the substrate that push the cell forward, squeezing it through the confined space, in a process termed ‘chimneying’ [17]. This is supported by findings in adhesion-deficient leukocytes that are nonetheless able to migrate efficiently [18].

Repolymerization

As expansion of the bleb begins to slow, cortical actin just beneath the bleb membrane begins to repolymerize. This mechanims of actin nucleation is unclear, as two of the most common actin nucleaters, the Arp2/3 complex and the formin mDia1, are not present beneath the membrane of filamin-deficient cells [13]. Studies have, however, shown the sequential recruitment of proteins to the bleb membrane, including F-actin, membrane-linker proteins, actin, actin-bundling proteins, and contractile proteins. The resultant bleb cytoskeleton underlying the membrane comprises multiple layers of F-actin tethered together by actin bundling proteins. During this process of repolymerization in the main cortical body of the bleb, F-actin at the base of the neck of the bleb disassembles due to actin turnover and redistribution [1].

Bleb Retraction

Retraction generally lasts between 60 to 120 seconds [1]. When the cells membrane lacks the stability afforded, for example, by cell-substrate contacts, the primary cascade involved in retraction is that of mysoin-RhoA-ROCK. In this cascade, myosin is recruited to the bleb lumen, GTP-loaded Rho activates ROCK, which directly phosphorylates myosin light chain and so generates actomyosin contraction .

This process can be regulated at multiple levels, both extracellularly and intracellularly, such as in the case of RhoA, its activity can be regulated by both extracellular signals and intracellular cascades. Indirect regulation of this cascade may also occur via members of the Rac subfamily of Rho GTPases, such as Rac1 or Dictyostelium discoideum RacB. The cascade can also be regulated through inhibition of its effector ROCK. In addition, the formins, Dia1 and mDia2 have been shown to be crucial in regulating retraction.

Circus Movement

Circus movements have been mainly observed in embryonic blastomeres, neuroectoderm and paraxial mesoderm, where a single bleb repeatedly propagates around the circumference of the cell with a period of 1–2 minutes. The cortical actin network reforms asymmetrically during the repolymerization phase of circus movements. This movement requires actomyosin contractility.

References

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