Molecular organization of integrin-based adhesion complexes in mouse Embryonic Stem Cells

Shumin Xia, Evelyn K.F. Yim, Pakorn Kanchanawong

Preprint posted on September 13, 2018


Superresolution architecture of pluripotency guarding adhesions

Aki Stubb, Camilo Guzmán, Elisa Närvä, Jesse Aaron, Teng-Leong Chew, Markku Saari, Mitro Miihkinen, Guillaume Jacquemet, Johanna Ivaska

Preprint posted on August 28, 2018

Stem cell focal adhesions are structurally different to those of differentiated cells and maintain pluripotency

Selected by Nicola Stevenson, Amanda Haage

Why This is Cool 

We have known for a while now that the microenvironment surrounding stem cells can direct their lineage specification1. Conversely, the microenvironment can also direct stem cells to retain pluripotency – the ability to differentiate into multiple lineages in the future2 – when differentiation is not required. In general, cells sense and respond to their microenvironment via integrin-mediated adhesion complexes termed focal adhesions (FAs) in 2D in vitro cell culture. What these two preprints do is provide extraordinary structural detail on how these FAs might differ in cells that maintain pluripotency versus those that are differentiated. Both studies utilize super-resolution microscopy techniques to study the nanoarchitecture of FAs – Xia et al. in mouse embryonic stem cells (mESCs) and Stubb & Guzmán et al. in human pluripotent stem cells (hPSCs). Both studies also compare their results with the known FA nanoarchitecture of adherent differentiated fibroblasts3. Although not directly comparable due to the use of different extracellular matrix substrates and stem cell origins, both studies agree that FA proteins are organized differently between stem cells and differentiated cells.


First, Xia et al. demonstrate that expression of integrin-mediated adhesion complex components (also called the adhesome) is similar between different kinds of pluripotent cells, but different when compared to differentiated cells. Particularly, the expression of LIM domain-containing proteins, which are known regulators of mechanotransduction, is lower in pluripotent cells. While the authors show that FAs from mESCs are mechanosensitive, they have attenuated responses. They also show that mESCs maintain pluripotency across various ECM substrates. The bulk of the study then focuses on the mESC FA structure. The authors find that these FAs have a hierarchical organization, similar to that seen in fibroblasts but with a few key differences. Firstly, talin appears to be more tightly packed in mESC FAs, with a reduced distance between the N- and C- terminus. Meanwhile, vinculin has an ECM-dependent localization, with an overall shift to being found closer to the membrane in mESC FAs.


Figure 1: Nanoscale Architecture of mESC FAs. Schematic diagram of the FA nanoscale architecture in mESCs, highlighting the core integrin-talin-actin connection (in red, yellow, blue, respectively). Vinculin is shown in green and marked by fluorescent protein probes at either the N- (blue) or C- (red) termini. The different configuration of vinculin in relation to integrin-talin-actin core is highlighted.



The study by Stubb & Guzmán et al. is a direct follow-up to their group’s previous work characterizing “cornerstone” FAs in hPSC colonies4. Here they show that these large FAs on the edge of hPSC colonies are connected to an actin fence, and altering cell morphology using nanopatterned substrates induces colony differentiation. They also characterize how cornerstone FA nanostructure differs from what has been reported for fibroblasts. For example, cornerstone FAs have different integrin subregions, with β5 and talin enriched at the edges, whilst β1 and αV have a homogeneous distribution across the adhesion. Talin, vinculin, and actin also show altered distributions within cornerstone FAs. Whilst talin is found closer to the membrane than normal and appears fully extended throughout the adhesion, vinculin is found in an alternative orientation with a cup-like distribution, which the authors suggest is indicative of a hyperactivated state. Actin forms not one, but two vertical layers still associated with actin regulating proteins. The functional significance of these differences remains largely unknown, but the authors do find that depleting vinculin has no impact on hPSC colony formation or pluripotency. Lastly, they investigate Kank 1 & 2 distribution. Kanks were recently identified as scaffold proteins that localize to FAs and are important for connecting FAs to microtubules5. Both Kank 1 & Kank 2 have unique distributions in cornerstone adhesions and interestingly their knockdown leads to smaller adhesions at the edge with an increase in adhesion size in the center of the colony.



Why We Selected Them 

Amanda – Understanding how pluripotency is maintained and how differentiation is specifically induced is essential for harnessing the power of stem cells for medical applications. Although it has classically been a large focus of cancer biology, it is now recognized that the environment in which all cells reside is not just a passive site of support. The chemical and physical properties of the environment provide instructional cues necessary for regulation. These studies provide the first in-depth look at how the proteins that sense and respond to the environment are organized differently based on maintenance of self-renewal vs. differentiation. It will be incredibly interesting to see the data that follows these studies, hopefully demonstrating the functional consequences to these changes in structure.


Nicola – These two studies both present beautifully visual evidence supporting a clear link between FA organisation and stem cell biology, which will no doubt be important when considering stem cell biology in development and disease in the future. Although these studies are not directly comparable due to the use of different extracellular matrix substrates and stem cell origins, there are substantial similarities between their findings which I found particularly interesting to see and made them worth highlighting. For example, stem cells from different sources exhibit similar adhesome expression profiles which do differ from the adhesome of differentiated cells. The adaptability of FA organisation is clearly important for both stem cell pluripotency and their mechanical properties and it will be interesting to see how future studies manage to translate these subtle changes into signalling outcomes and relate the findings to in vivo situations.


Open Questions 

  1. Both studies highlight vinculin as a FA component with altered distributions in stem versus differentiated cells, yet Stubb & Guzmán et al. show it is not required to maintain pluripotency. This raises the question what is the relevance of the altered position of vinculin within the FA of stem cells to stem cell biology?
  2. If not vinculin, then which FA components are essential for maintaining pluripotency?
  3. How does silencing of Kank1 and Kank2 affect pluripotency?
  4. How do stem cell FAs react to adhesion on a more physiologically relevant cell derived matrix that more closely recapitulates the stem cell niche in vivo and how are they structured in situ?
  5. Do the FAs present in the centre of the hPSC cell colonies studied by Stubb & Guzmán et al. more closely resemble those studied in mESC by Xia et al. than the cornerstone adhesions?


Related References

  1. Microenvironment directs stem cell differentiation
    1. Engler AJ., Sen S., Sweeny HL., and Discher DE. Matrix elasticity directs stem cell lineage specification. (2006). 126(4):677-89.
  2. Microenvironment is fundamental to maintaining pluripotency
    1. Braam SR., Zeinstra L., Litjens S., Ward-van Oostwaard D., van den Brink S., van Laake L., Lebrin F., Kats P., Hochstenbach R., Passier R., Sonnenberg A., and Mummery CL. Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin. Stem Cells. (2008). 26(9):2257-65.
  3. FA organization in fibroblasts
    1. Kanchanawong P., Shtengel G., Pasapera AM., Ramko EB., Davidson MW., Hess HF., and Waterman CM. Nanoscale architecture of integrin-based cell adhesions. Nature. (2010). 468(7323):580-584.
  4. Cornerstone FAs in hPSCs.
    1. Narva E., Stubb A., Guzman C., Blomqvist M., Balboa D., Lerche M., Saari M., Otonkoski T., and Ivaska J. A strong contractile actin fence and large adhesions direct human pluripotent colony morphology and adhesion. Stem Cell Reports. (2017). 9(1):67-76.
  5. Kank in FAs

Boucht BP., Gough RE., Ammon YC., Van de Willige D., Post H., Jacquemet G., Altelaar AFM., Heck AJR., and Goult BT. Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions.


Posted on: 2nd November 2018

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