Superresolution microscopy

Fluorescence microscopy provides exquisite sensitivity and superb specificity for detecting and visualizing molecules in cells. However, the resolution of light microscope is conventionally limited by diffraction to, at best, ~250 nm. Visualization of cellular structures at the nanoscale level has long been dependent on electron microscopy (EM). However, EM is generally hampered by poor molecular specificity, laborious sample preparation, incompatibility with live/hydrated specimens, and limited multichannel capability. The aim of super-resolution microscopy is therefore to combine the molecular specificity and sensitivity afforded by fluorescence with nanometer-scale spatial resolution—in other words, ligh
t-based imaging with a
resolving power comparable to EM. 

A major focus of our research group is the development and application of super-resolution microscopy. Our group operates an iPALM (interferometric PhotoActivated Localization Microscopy) 3-D single-molecule superresolution microscopes at MBI (Shtengel et al., PNAS 2009). We continue to develop this technique as well as other advanced imaging methods, applying these technologies in conjunction with molecular and cell biological approaches to gain mechanistic insights into cellular mechanotransduction and other cell biological processes.

Examples of super-resolution images from our group can be found here

    
(Shtengel et al., PNAS 2009)                                                               (Kanchanawong et al., Methods Mol Biol 2013)                                                                (Shtengel et al., Methods Cell Biol. 2014)

Integrin- & cadherin-mediated cell adhesions

Cells in our bodies constantly interact with their neighbors and their environments. These cell-cell and cell-matrix interactions are inter-dependent processes essential for all facets of well-being: tissue integrity, proper developmental morphogenesis, immunity, wound healing, and many other homeostatic balances. Defects in these processes are involved in serious conditions ranging from developmental defects to cancer metastasis. Several receptors mediate cell-cell and cell-matrix interactions, forming complexes with signaling and/or structural and mechanical roles. We’re interested in the integrin- and cadherin-based complexes (involved in cell-matrix and cell-cell interactions, respectively), particularly in their roles in cellular mechanotransduction.

Mechanotransduction refers to the conversion of mechanical stimuli into biochemical signals and encompass multiple processes. The integrin-based complexes (also known as Focal
Adhesions) are coupled to the bundled actin stress fibers and are among the most well-documented organelles responsible for the sensing of the mechanical microenvironment—mechanical compliance (stiffness) as well as the topography (texture) of the matrix. Focal Adhesions generally respond to stiff substrate by enlarging—in terms of both the size of the focal adhesions complexes and the associated actin stress fiber—in concert with the increase in myosin II-mediated contractility. Thus, Focal Adhesions are distinctly mechanosensitive, actively adjusting their sizes in response to changes in the stiffness and intracellular tension. Of great importance, mechano-signaling from integrins and associated Focal Adhesions proteins are implicated in invasive metastasis, as well as in the determination of cell fate during stem cell differentiation. Thus, insights into the molecular mechanisms of Focal Adhesions mechanotransduction are both fundamentally important and biomedically relevant. We have earlier applied iPALM to reveal, for the first time, the nanoscale architecture of the Focal Adhesions (Kanchanawong et al., Nature 2010). Our current research focuses on investigating the molecular basis of the Focal Adhesions architecture, as well as the roles of Focal Adhesions in stem cell mechanobiology.

In recent years, mechanotransduction by cadherin-based cell-cell adhesions have been increasingly recognized. Among the protein components of the cell-cell junctions, some are unique but several are also common to Focal Adhesions. Likewise, cell-cell and cell-matrix interactions are known to exhibit interesting and complicated crosstalks. Compared to Focal Adhesions, there remain several molecular and structural aspects of the cell-cell junctions that are not as well characterized. To further the understanding of this structure, we’re employing superresolution microscopy techniques to decipher how proteins are organized within cadherin-based complexes at the molecular scale.

                
(Kanchanawong et al., Nature 2010)                    (Wu et al., Developmental Cell 2015)

Nanoscale architecture and cellular functions

At the molecular scale, the structure-function paradigm has been one of the key guiding principles in biology. The structures of the proteins are dictated by the amino-acid sequences which determine the shape of the folded proteins, their stability, their flexibility/rigidity, as well as the complementarity with other ligands or protein in binding interactions. In parallel, at the anatomical scale, the “Forms follow Functions” principle is widely appreciated and ubiquitously documented. However, our understanding of the structure-function relationship at the nano/mesoscale (10-200 nm) has long been very sparse.

Technological bottlenecks have long limited our understanding of biological systems at such nano/mesoscale. This length scale is too large for structural biology techniques but too small for light microscopy, which is unable to resolve features smaller than ~250 nm due to the diffraction limit. At the same time, limitations in many aspects of EM restrict the ability to comprehend molecular complexity at this length scale. Nevertheless, the nano/mesoscale is highly pertinent for cellular processes, as this is the length scale at which complex molecular machines are organized. With the development of superresolution microscopy, this length scale has recently been opened up for fluorescence-based imaging. We have been among the first groups to exploit this technology to decipher the nanoscale architecture of a complex protein-based machine, the Focal Adhesions (Kanchanawong et al., Nature 2010).
Our iPALM study revealed a multilaminar architecture in which proteins are stratified along the axial dimension into partially overlapping zones that we termed: membrane proximal layer, force transduction layer, and actin regulatory layer.

Focal adhesions can be said to epitomize a highly complex molecular assembly that perform diverse yet tightly integrated cellular functions. Due to its intrinsic mechanical functions and its close association with the actin cytoskeleton, the nanoscale architecture of the focal adhesions can be expected to strongly influence their physical properties, though these remain not well understood. Furthermore, how the interplay between the nanoscale compartmentalization, clustering, molecular crowding, and diffusion influence dynamic processes such as Focal Adhesion assembly /maturation/turnover, signalling actuated by adhesion cues or mechanical cues, and regulation of biophysical roles such as traction force transmission remains to be explored. A major area of our research is focused on interrogating the structure-function relationship in Focal Adhesions by combining superresolution techniques with cellular/molecular perturbations. We anticipate that insights from the Focal Adhesions systems may also be instructive for other complex protein-based machines which may not be as experimentally tractable.

Beyond the Focal Adhesions and cadherin-based complexes, we also are applying super-resolution microscopy to visualize several other subcellular structures in collaboration with researchers both in Singapore and overseas.

(Nomachi et al. PlosONE 2013)