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Increased asymmetric and multi-daughter cell division in mechanically confined microenvironments.

ABSTRACT: As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows controllable variation in the level of confinement in a single axis allowing observation of cell growth and division at the single-cell level. The device is capable of creating precise confinement conditions in the vertical direction varying from high (3 µm) to low (7 µm) confinement while also varying the substrate stiffness (E = 130 kPa and 1 MPa). The Human cervical carcinoma (HeLa) model with a known 3N+ karyotype was used for this study. For this cell line, we observe that mechanically confined cell cycles resulted in stressed cell divisions: (i) delayed mitosis, (ii) multi- daughter mitosis events (from 3 up to 5 daughter cells), (iii) unevenly sized daughter cells, and (iv) induction of cell death. In the highest confined conditions, the frequency of divisions producing more than two progeny was increased an astounding 50-fold from unconfined environments, representing about one half of all successful mitotic events. Notably, the majority of daughter cells resulting from multipolar divisions were viable after cytokinesis and, perhaps suggesting another regulatory checkpoint in the cell cycle, were in some cases observed to re-fuse with neighboring cells post-cytokinesis. The higher instances of abnormal mitosis that we report in confined mechanically stiff spaces, may lead to increased rates of abnormal, viable, cells in the population. This work provides support to a hypothesis that environmental mechanical cues influences structural mechanisms of mitosis such as geometric orientation of the mitotic plane or planes.

SUBMITTER: Tse HT

PROVIDER: S-EPMC3382600 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Increased asymmetric and multi-daughter cell division in mechanically confined microenvironments.

Tse Henry Tat Kwong HT Weaver Westbrook McConnell WM Di Carlo Dino D

PloS one 20120625 6

As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows contr ...[more]

PMID: 22761717

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Project description:UnlabelledDividing cells of the coccoid Gram-positive bacterium Staphylococcus aureus undergo extremely rapid (millisecond) daughter cell separation (DCS) driven by mechanical crack propagation, a strategy that is very distinct from the gradual, enzymatically driven cell wall remodeling process that has been well described in several rod-shaped model bacteria. To determine if other bacteria, especially those in the same phylum (Firmicutes) or with similar coccoid shapes as S. aureus, might use a similar mechanically driven strategy for DCS, we used high-resolution video microscopy to examine cytokinesis in a phylogenetically wide range of species with various cell shapes and sizes. We found that fast mechanically driven DCS is rather rare in the Firmicutes (low G+C Gram positives), observed only in Staphylococcus and its closest coccoid relatives in the Macrococcus genus, and we did not observe this division strategy among the Gram-negative Proteobacteria In contrast, several members of the high-G+C Gram-positive phylum Actinobacteria (Micrococcus luteus, Brachybacterium faecium, Corynebacterium glutamicum, and Mycobacterium smegmatis) with diverse shapes ranging from coccoid to rod all undergo fast mechanical DCS during cell division. Most intriguingly, similar fast mechanical DCS was also observed during the sporulation of the actinobacterium Streptomyces venezuelaeImportanceMuch of our knowledge on bacterial cytokinesis comes from studying rod-shaped model organisms such as Escherichia coli and Bacillus subtilis Less is known about variations in this process among different bacterial species. While cell division in many bacteria has been characterized to some extent genetically or biochemically, few species have been examined using video microscopy to uncover the kinetics of cytokinesis and daughter cell separation (DCS). In this work, we found that fast (millisecond) DCS is exhibited by species in two independent clades of Gram-positive bacteria and is particularly prevalent among the Actinobacteria, a diverse group that includes significant pathogens as well as bacteria that generate medically important antibiotics.

Project description:Asymmetric cell division is common in biology and plays critical roles in differentiation and development. Unicellular organisms are often used as model systems for understanding the origins and consequences of asymmetry during cell division. Although basic as compared to mammalian cells, these are already quite complex. We report complete budding and asymmetric fission of very simple nonliving model cells to produce daughter vesicles that are chemically distinct in both interior and membrane compositions. Our model cells are based on giant lipid vesicles (GVs, 10-30 μm) encapsulating a polyethylene glycol (PEG)/dextran aqueous two-phase system (ATPS) as a crowded and compartmentalized cytoplasm mimic. Ternary lipid compositions were used to provide coexisting micrometer-scale liquid disordered (L(d)) and liquid ordered (L(o)) domains in the membranes. ATPS-containing vesicles formed buds when sucrose was added externally to provide increased osmotic pressure, such that they became not only morphologically asymmetric but also asymmetric in both their interior and their membrane compositions. Further increases in osmolality drove formation of two chemically distinct daughter vesicles, which were in some cases connected by a lipid nanotube (complete budding), and in others were not (fission). In all cases, separation occurred at the aqueous-aqueous phase boundary, such that one daughter vesicle contained the PEG-rich aqueous phase and the other contained the dextran-rich aqueous phase. PEGylated lipids localized in the L(o) domain resulted in this membrane domain preferentially coating the PEG-rich bud prior to division, and subsequently the PEG-rich daughter vesicle. Varying the mole ratio of lipids resulted in excess surface area of L(o) or L(d) membrane domains such that, upon division, this excess portion was inherited by one of the daughter vesicles. In some cases, a second "generation" of aqueous phase separation and budding could be induced in these daughter vesicles. Asymmetric fission of a simple self-assembled model cell, with production of daughter vesicles that harbored different protein concentrations and lipid compositions, is an example of the seemingly complex behavior possible for simple molecular assemblies. These compartmentalized and asymmetrically dividing ATPS-containing GVs could serve as a test bed for investigating possible roles for spatial and organizational cues in asymmetric cell division and inheritance.

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Increased asymmetric and multi-daughter cell division in mechanically confined microenvironments.

Publications

Increased asymmetric and multi-daughter cell division in mechanically confined microenvironments.

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