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Cytoskeletal Disassembly and Cell Rounding Promotes Adipogenesis from ES Cells

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Abstract

Biomechanical signals such as cell shape and spreading play an important role in controlling stem cell commitment. Cell shape, adhesion and spreading are also affected by calreticulin, a multifunctional calcium-binding protein, which influences several cellular processes, including adipogenesis. Here we show that cytoskeletal disruption in mouse embryonic stem cells using cytochalasin D or nocodazole promotes adipogenesis. While cytochalasin D disrupts stress fibres and inhibits focal adhesion formation, nocodazole depolymerises microtubules and promotes focal adhesion formation. Furthermore, cytochalasin D increases the levels of both total and activated calcium/calmodulin-dependent protein kinase II, whereas nocodazole decreases it. Nevertheless, both treatments significantly increase the adipogenic potential of embryonic stem cells in vitro. Both cytochalasin D and nocodazole exposure caused cell rounding suggesting that it is cell shape that causes the switch towards the adipogenic programme. Calreticulin-containing embryonic stem cells, under baseline conditions, show low adipogenic potential, have low activity of signalling via calcium/calmodulin-dependent protein kinase II and display normal adhesive properties and cellular spreading in comparison to the highly adipogenic but poorly spread calreticulin-deficient ES cells. We conclude that forced cell rounding via cytoskeletal disruption overrides the effects of calreticulin, an ER chaperone, thus negatively regulating adipogenesis via focal adhesion-mediated cell spreading.

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Abbreviations

C/EBP:

CCAAT-enhancer binding protein

CaMKII:

Ca2+/calmodulin-dependent protein kinase II

EB:

embryoid body

EGTA:

ethylene glycol tetraacetic acid

ER:

endoplasmic reticulum

ES cells:

embryonic stem cells

GAPDH:

glyceraldehde 3-phosphate dehydrogenase

PBS:

Phosphate-buffered saline

PIPES:

Piperazine-1,4-bis(2-ethanesulfonic acid)

PPAR:

peroxisome proliferator activated receptor

SDS PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

WT:

wild type

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Acknowledgments

We are grateful to Dr. Marek Michalak for his generosity with anti-calreticulin antibodies and to Dr. Sylvia Papp for critical reading of the manuscript. M.O. is a member of the Heart & Stroke/Richard Lewar Centre of Excellence. This work was supported by grants from CIHR (MPO-36384) and from the Heart and Stroke Foundation of Ontario (T 6181).

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Author notes

  1. Eva Szabo

    Present address: McMaster Stem Cell and Cancer Research Institute, McMaster University, Hamilton, ON, L9G 4L6, Canada

Authors and Affiliations

  1. Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King’s College Circle, Medical Sciences Building, room 6326, Toronto, ON, M5S 1A8, Canada

    Tianshu Feng, Eva Szabo, Ewa Dziak & Michal Opas

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Correspondence to Michal Opas.

Additional information

T. Feng: Collection and assembly of data, Data analysis and interpretation, Manuscript writing E. Szabo: Collection and assembly of data, Data analysis and interpretation E. Dziak: Collection of data M. Opas: Conception and design, Data analysis and interpretation, Manuscript writing and Final approval

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Supplemental Fig. 1

Cytochalasin D-treated cells contain an intact microtubule network. This composite shows three confocal sections taken through the top, middle and bottom of a cytochalasin D-treated WT ES cell clump. Intact microtubules can be detected in most of the cells, especially in the large cell endowed with a protrusion. Scale divisions: 10, 50 and 100 μm. (GIF 52 kb)

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Feng, T., Szabo, E., Dziak, E. et al. Cytoskeletal Disassembly and Cell Rounding Promotes Adipogenesis from ES Cells. Stem Cell Rev and Rep 6, 74–85 (2010). https://doi.org/10.1007/s12015-010-9115-8

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