Microtubule-Mediated Inositol Lipid Signaling Plays Critical Roles in Regulation of Blebbing

Abstract

Cells migrate by extending pseudopods such as lamellipodia and blebs. Although the signals leading to lamellipodia extension have been extensively investigated, those for bleb extension remain unclear. Here, we investigated signals for blebbing in Dictyostelium cells using a newly developed assay to induce blebbing. When cells were cut into two pieces with a microneedle, the anucleate fragments vigorously extended blebs. This assay enabled us to induce blebbing reproducibly, and analyses of knockout mutants and specific inhibitors identified candidate molecules that regulate blebbing. Blebs were also induced in anucleate fragments of leukocytes, indicating that this assay is generally applicable to animal cells. After cutting, microtubules in the anucleate fragments promptly depolymerized, followed by the extension of blebs. Furthermore, when intact cells were treated with a microtubule inhibitor, they frequently extended blebs. The depolymerization of microtubules induced the delocalization of inositol lipid phosphatidylinositol 3,4,5-trisphosphate from the cell membrane. PI3 kinase-null cells frequently extended blebs, whereas PTEN-null cells extended fewer blebs. From these observations, we propose a model in which microtubules play a critical role in bleb regulation via inositol lipid metabolism.

Shear force-based genetic screen reveals negative regulators of cell adhesion and protrusive activity

“…It will be interesting to learn how, for example, proteins localized to the cytosol, plasma membrane, MTOC, or the nucleus can all negatively regulate PI3K pathways. Two of the RAM proteins localizing to the MTOC (SepA and DSPA) provide further support for the importance of microtubules in regulating cell polarity, migration, and adhesion (28,(79)(80)(81). Moreover, almost all of the phenotypes can be suppressed by overexpression of dominant negative Rap1 which is thought to be a regulator in multiple pathways involved in chemotaxis.…”

Cell ingression: Relevance to limb development and for adaptive evolution

“…Consistent with these observations, pharmacological inhibition of Myosin II and rho-associated protein kinase decreased neural crest cell blebbing and delamination (Berndt et al, 2008). Other experiments show that Myosin II function is required to promote bleb formation among gastrula stage mesodermal cells in zebrafish (Weiser et al, 2009) and in Dictyostelium (Sugiyama, Pramanik, & Yumura, 2015), presumably by increasing cortical tension and cytosolic hydrostatic pressure (Tinevez et al, 2009). Bleb formation in Dictyostelium is attributable to the focal disassembly of microtubules that likely stabilize membrane phosphatidylinositol 3,4,5-trisphosphate, a positive regulator of actin assembly (Sugiyama et al, 2015).…”

“…Other experiments show that Myosin II function is required to promote bleb formation among gastrula stage mesodermal cells in zebrafish (Weiser et al, 2009) and in Dictyostelium (Sugiyama, Pramanik, & Yumura, 2015), presumably by increasing cortical tension and cytosolic hydrostatic pressure (Tinevez et al, 2009). Bleb formation in Dictyostelium is attributable to the focal disassembly of microtubules that likely stabilize membrane phosphatidylinositol 3,4,5-trisphosphate, a positive regulator of actin assembly (Sugiyama et al, 2015). Myosin phosphatase limits the duration of blebbing among mesodermal cells in zebrafish, thereby allowing transition to intercalative behaviors (Weiser et al, 2009).…”

Involvement of β- and γ-actin isoforms in actin cytoskeleton organization and migration abilities of bleb-forming human colon cancer cells

“…It was shown that bleb formation is critically dependent on the level of myosin contractility [59]. It also contributes to the extension of blebs by increasing intracellular pressure [86]. Myosin II recruitment to the bleb is thought to drive bleb retraction.…”