Microinjection of the catalytic fragment of myosin light chain kinase into dividing cells: effects on mitosis and cytokinesis.

Fishkind, Douglas J.; Cao, Long-Guang; Wang, Y.-L.

doi:10.1083/jcb.114.5.967

Cited by 95 publications

(78 citation statements)

References 66 publications

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“…The myosin II contractile activity is stimulated through phosphorylation of myosin light chain on serine 19 by MLCK which catalyzes the interaction between myosin and actin needed to produce sliding forces for cell contraction and movement (59,60). Microinjection of catalytically active MLCK can induce bleb formation (41). In addition, the small GTPases Rho, Rac and CDC42 [which are elevated in pII cells (3)] play an important role in cytoskeletal rearrangement and actin stress fiber formation (61,62); treatment with the actin de-polymerizer cytochalasin-D, or inhibitors of MLCK and Rho kinase activity, all inhibited bleb formation in serum-deprived z-VAD-FMK treated PC6-3 cells (51).…”

Section: Discussionmentioning

confidence: 99%

“…The alkaline induced shrinking and rounding phenomenon of the cell that we documented previously, when examined more closely at higher magnification, indicates thickening of the plasma membrane (often also referred to as ruffling) and formation of vesicular invaginations closely resembling cytoplasmic 'blebbing' described during early embryogenic migration (38). In a more general context, and as distinct from the more extensively studied membrane protrusions enabling motile functions such as lamellipodia and filopodia, blebs are associated with motility (39), cell spreading (40), cytokinesis (41) and mitosis (42)(43)(44)(45).…”

Section: Discussionmentioning

confidence: 99%

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Bleb formation is induced by alkaline but not acidic pH in estrogen receptor silenced breast cancer cells

Khajah

Mathew

Alam-Eldin

et al. 2015

International Journal of Oncology

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Abstract.De novo and acquired resistance to endocrinebased therapies in breast cancer occurs in parallel with epithelial to mesenchymal transition (EMT), which is associated with enhanced proliferative and metastatic potential, and poor clinical outcome. We have established several endocrine insensitive breast cancer lines by shRNA-induced depletion of estrogen receptor (ER) by transfection of MCF7 cells. All of these exhibit EMT. We have previously reported that brief exposure of specifically ER -breast cancer cells, to extracellular alkaline pH, results in cell rounding and segregation, and leads to enhanced invasive potential. In this study we describe more detailed morphological changes and compare these with cell exposure to acidic pH. Morphological changes and localization of various molecules critical for cell adhesion and motility, associated with pH effects, were assessed by live cell microscopy, electron microscopy, and immunofluorescence. Exposure of either ER -or ER + breast cancer cells to extracellular acidic pH did not induce significant changes in morphological appearance. Conversely, brief exposure of specifically ER silenced cells, to alkaline pH, resulted in cell contractolation and formation of bleb-like actin-rich structures which were evenly distributed on the outer membrane. Integrin α2, FAK, and JAM-1 were found in the cytoplasm streaming into the newly formed blebs. These blebs appear to be related to cell polarity and movement. Pre-treatment with cytochalasin-D or inhibitors of Rho or MLCK prevented both contractolation and bleb formation. Our data suggest that the effect of pH on the microenvironment of endocrine resistant breast cancer cells needs to be more extensively investigated. Alkaline, rather than acidic pH, appears to induce dramatic morphological changes, and enhances their invasive capabilities, through re-organization of cortical actin.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Bleb formation is induced by alkaline but not acidic pH in estrogen receptor silenced breast cancer cells

Khajah

Mathew

Alam-Eldin

et al. 2015

International Journal of Oncology

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“…We were drawn to consider poroelasticity by analysis of blebbing [12], a dramatic type of motility that many animal cells display when they divide [30,31] or undergo apoptosis [32]. Blebbing is typically a cyclic phenomenon where a bleb nucleates, expands, and contracts over 1-3 minutes without moving laterally ( figure 5 [33,34]).…”

Section: Exhibit 1: Blebbing As a Window Into Cell Hydraulicsmentioning

confidence: 99%

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

Mitchison

Charras

Mahadevan

2008

Seminars in Cell & Developmental Biology

143

113

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Two views have dominated recent discussions of the physical basis of cell shape change during migration and division of animal cells: the cytoplasm can be modeled as a viscoelastic continuum, and the forces that change its shape are generated only by actin polymerization and actomyosin contractility in the cell cortex. Here, we question both views: we suggest that the cytoplasm is better described as poroelastic, and that hydrodynamic forces may be generally important for its shape dynamics. In the poroelastic view, the cytoplasm consists of a porous, elastic solid (cytoskeleton, organelles, ribosomes) penetrated by an interstitial fluid (cytosol) that moves through the pores in response to pressure gradients. If the pore size is small (30-60nm), as has been observed in some cells, pressure does not globally equilibrate on time and length scales relevant to cell motility. Pressure differences across the plasma membrane drive blebbing, and potentially other type of protrusive motility. In the poroelastic view, these pressures can be higher in one part of a cell than another, and can thus cause local shape change. Local pressure transients could be generated by actomyosin contractility, or by local activation of osmogenic ion transporters in the plasma membrane. We propose that local activation of Na+/H+ antiporters (NHE1) at the front of migrating cells promotes local swelling there to help drive protrusive motility, acting in combination with actin polymerization. Local shrinking at the equator of dividing cells may similarly help drive invagination during cytokinesis, acting in combination with actomyosin contractility. Testing these hypotheses is not easy, as water is a difficult analyte to track, and will require a joint effort of the cytoskeleton and ion physiology communities.

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“…In fact, surface blebbing can be dramatically extended (up to several days) if caspases are inhibited (Mills et al, 1998b), cells eventually dying by a caspase-independent, non-apoptotic form of cell death (McCarthy et al, 1997). Moreover, viable cells also display myosin II-dependent plasma membrane blebbing when spreading, migrating, dividing, or when under conditions of stress (Bereiter-Hahn et al, 1990;Cunningham, 1995;Fishkind et al, 1991;Huot et al, 1998;Straight et al, 2003). Taken together, these observations raise the possibility that apoptotic blebbing is simply a by-product of cellular reorganisation, perhaps linked to the partial loss of cellular contact with the substratum, and does not play a direct role in apoptotic progression.…”

Section: Introductionmentioning

confidence: 99%

Active relocation of chromatin and endoplasmic reticulum into blebs in late apoptotic cells

Lane

Allan

Woodman

2005

Journal of Cell Science

130

110

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Plasma membrane blebbing is a defining characteristic of apoptosis, but its significance is not understood. Using live-cell imaging we have identified two phases of apoptotic blebbing. The early phase is restricted to adherent cells, and is prevented by the Rho-activated kinase inhibitor Y27632. The late phase is partially resistant to Y27632, and generates morphologically distinct membrane protrusions that are likely precursors to apoptotic bodies. Late blebbing is observed in all apoptotic cells tested. It occurs at a fixed period before phosphatidyl serine exposure, indicating that it is a universal and important feature of apoptosis. Late blebs contain a cortical layer of endoplasmic reticulum that often surrounds condensed chromatin, while other organelles are excluded. The appearance in some apoptotic cells of partially formed sheets of endoplasmic reticulum suggest that these cortical layers are newly formed by the remodelling of the endoplasmic reticulum of interphase cells. Formation of endoplasmic reticulum and chromatin-containing blebs requires both actin and microtubules, and is prevented by the caspase-6 inhibitor zVEID.fmk.

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Microinjection of the catalytic fragment of myosin light chain kinase into dividing cells: effects on mitosis and cytokinesis.

Cited by 95 publications

References 66 publications

Bleb formation is induced by alkaline but not acidic pH in estrogen receptor silenced breast cancer cells

Bleb formation is induced by alkaline but not acidic pH in estrogen receptor silenced breast cancer cells

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

Active relocation of chromatin and endoplasmic reticulum into blebs in late apoptotic cells

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