Structured illumination microscopy reveals focal adhesions are composed of linear subunits

The ability to mechanically interact with the extracellular matrix is a fundamental feature of adherent eukaryotic cells. Cell–matrix adhesion in many cell types is mediated by protein complexes called focal adhesions (FAs). Recent progress in super resolution microscopy revealed FAs possess an internal organization, yet such methods do not enable observation of the formation and dynamics of their internal structure in living cells. Here, we combine structured illumination microscopy (SIM) with total internal reflection fluorescence microscopy (TIRF) to show that the proteins inside FA patches are distributed along elongated subunits, typically 300 ± 100 nm wide, separated by 400 ± 100 nm, and individually connected to actin cables. We further show that the formation and dynamics of these linear subunits are intimately linked to radial actin fiber formation and actomyosin contractility. We found FA growth to be the result of nucleation of new linear subunits and their coordinated elongation. Taken together, this study reveals that the basic units of mature focal adhesion are 300‐nm‐wide elongated, dynamic structures. We anticipate this ultrastructure to be relevant to investigation of the function of FAs and their behavior in response to mechanical stress. © 2015 Wiley Periodicals, Inc.

[1]  B. Geiger,et al.  The integrin adhesome: from genes and proteins to human disease , 2014, Nature Reviews Molecular Cell Biology.

[2]  Eric W. Frey,et al.  Multiscale mechanobiology: mechanics at the molecular, cellular, and tissue levels , 2013, Cell & Bioscience.

[3]  Ning Wang,et al.  Mechanotransduction at focal adhesions: from physiology to cancer development , 2013, Journal of cellular and molecular medicine.

[4]  Keiji Naruse,et al.  Rac1 Recruitment to the Archipelago Structure of the Focal Adhesion through the Fluid Membrane as Revealed by Single-Molecule Analysis , 2013, Cytoskeleton.

[5]  T. Fujiwara,et al.  Archipelago architecture of the focal adhesion: Membrane molecules freely enter and exit from the focal adhesion zone , 2012, Cytoskeleton.

[6]  Léa Trichet,et al.  Evidence of a large-scale mechanosensing mechanism for cellular adaptation to substrate stiffness , 2012, Proceedings of the National Academy of Sciences.

[7]  Z. Kam,et al.  Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing , 2011, Nature Cell Biology.

[8]  Thomas Boudou,et al.  A hitchhiker's guide to mechanobiology. , 2011, Developmental cell.

[9]  Patrick W Oakes,et al.  Spatiotemporal constraints on the force-dependent growth of focal adhesions. , 2011, Biophysical journal.

[10]  Michael W. Davidson,et al.  Nanoscale architecture of integrin-based cell adhesions , 2010, Nature.

[11]  Benjamin Geiger,et al.  Dissecting the molecular architecture of integrin adhesion sites by cryo-electron tomography , 2010, Nature Cell Biology.

[12]  Miguel Vicente-Manzanares,et al.  Non-muscle myosin II takes centre stage in cell adhesion and migration , 2009, Nature Reviews Molecular Cell Biology.

[13]  B. Geiger,et al.  The heel and toe of the cell's foot: a multifaceted approach for understanding the structure and dynamics of focal adhesions. , 2009, Cell motility and the cytoskeleton.

[14]  Gaudenz Danuser,et al.  Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed , 2008, The Journal of cell biology.

[15]  Jean-Jacques Meister,et al.  Comparative Dynamics of Retrograde Actin Flow and Focal Adhesions: Formation of Nascent Adhesions Triggers Transition from Fast to Slow Flow , 2008, PloS one.

[16]  Michael W. Davidson,et al.  Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes , 2007, Proceedings of the National Academy of Sciences.

[17]  Yu-Li Wang,et al.  Retrograde fluxes of focal adhesion proteins in response to cell migration and mechanical signals. , 2007, Molecular biology of the cell.

[18]  S. Itzkovitz,et al.  Functional atlas of the integrin adhesome , 2007, Nature Cell Biology.

[19]  Colin K. Choi,et al.  Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells , 2007, The Journal of cell biology.

[20]  Gaudenz Danuser,et al.  Differential Transmission of Actin Motion Within Focal Adhesions , 2007, Science.

[21]  Viola Vogel,et al.  Mechanotransduction involving multimodular proteins: converting force into biochemical signals. , 2006, Annual review of biophysics and biomolecular structure.

[22]  M. Sheetz,et al.  Local force and geometry sensing regulate cell functions , 2006, Nature Reviews Molecular Cell Biology.

[23]  P. Janmey,et al.  Tissue Cells Feel and Respond to the Stiffness of Their Substrate , 2005, Science.

[24]  Daniel J Müller,et al.  Analyzing focal adhesion structure by atomic force microscopy , 2005, Journal of Cell Science.

[25]  N. Balaban,et al.  Adhesion-dependent cell mechanosensitivity. , 2003, Annual review of cell and developmental biology.

[26]  Benjamin Geiger,et al.  Focal Contacts as Mechanosensors Externally Applied Local Mechanical Force Induces Growth of Focal Contacts by an Mdia1-Dependent and Rock-Independent Mechanism , 2001 .

[27]  M. Gustafsson Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy , 2000, Journal of microscopy.

[28]  L. Smilenov,et al.  Focal adhesion motility revealed in stationary fibroblasts. , 1999, Science.

[29]  Y. Wang,et al.  Cell locomotion and focal adhesions are regulated by substrate flexibility. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Daniel Choquet,et al.  Extracellular Matrix Rigidity Causes Strengthening of Integrin–Cytoskeleton Linkages , 1997, Cell.

[31]  M. Piel,et al.  Protein micropatterns: A direct printing protocol using deep UVs. , 2010, Methods in cell biology.

[32]  B. Geiger,et al.  Environmental sensing through focal adhesions , 2009, Nature Reviews Molecular Cell Biology.

[33]  R. Kessin,et al.  Dictyostelium: Cell Motility and the Cytoskeleton , 2001 .