Unsolved Problems in Cell Biology: Why Do Microvilli Translocate?

Ligand sensing structures like microvilli translocate in the plane of the plasma membrane. What is the functional significance of this and what are the mechanisms of force production and structural integrity?

George M. Langford

Many different types of cells in the intact organism elaborate cell membrane protrusions for a variety of functions. Neuronal cells, for example, produce mushroom-shaped spines that function as sites of synapse formation on the surfaces of the dendrites. Pancreatic beta cells and T cells produce microvilli that function as sites of ligand sensing. These tubular protrusions contain a dense core of actin filaments that function to generate force and maintain the membrane deformation. In addition, these tubular membrane compartments contain specific protein complexes involved in signaling cascades that serve to initiate downstream processes such as long-term potentiation in neurons and glucose-stimulated insulin secretion in pancreatic beta cells.
Interestingly, in most cell types, these membrane protrusions have been shown to be motile, i.e., they have the ability to translocate in the plane of the membrane. In the case of microvilli on pancreatic beta cells, the movement is directed and occurs over distances that are several times the diameter of individual microvilli.1 These observations raise important questions: What is the functional significance of this motility, what is the mechanism of movement, and how is structural integrity of microvilli maintained?
These questions touch on a variety of important problems in cell biology including cytoskeletal–membrane dynamics and ligand sensing at the cellular level. The microvillus represents a structural unit that has the ability to translocate in the fluid lipid bilayer. Membrane fluidity at the junction between the microvillus and the plasma membrane becomes important for movement of the microvillus as an integral unit. These unanswered questions have profound implications on how we understand the sensory function of structures like microvilli and how we design studies to understand complex diseases like type 2 diabetes.
Why are these structures motile? One obvious answer is to improve search efficiency for ligands or binding partners that may exist at low concentrations. Microvilli on rat islet beta cells are hot spots for GLUT2,2 the high-Km glucose facilitative transporter, and therefore represent the primary sites for glucose sensing and uptake. To definitively demonstrate that movement represents an efficient search strategy for ligands, one has to record the dynamics of these structures in real time in living cells at sufficient spatial and temporal resolution and with multicolor fluorescent markers of membrane and cytoskeletal components to capture the membrane and cytoskeletal dynamics.
Imaging techniques are improving at a rapid rate, but a significant gap exists between the super resolution (SR) techniques that rely on fixed specimen and those that have the ability to image living cells in real time. Total internal reflection fluorescence structured illumination microscopy (TIRF-SIM) is one SR technique that has the advantage of imaging live cells far faster and with orders of magnitude less light than is required for other forms of SR fluorescence microscopy. However, the resolution achieved with TIRF-SIM is limited to a 2-fold gain beyond a conventional fluorescence microscope, or ~100 nm with visible light. This improvement in resolution is sufficient to image microvilli that average 150 nm in diameter, a size that is difficult to detect without SR capability. Nevertheless, the spatio-temporal resolution of TIRF-SIM is not sufficient to capture the full range of dynamic changes that occur in the subsecond time frame and in the sub-100-nm size domain.
Betzig and colleagues developed a techniques called patterned activation nonlinear SIM (PA NL-SIM) that extends TIRF-SIM to the sub-100-nm spatial domain by exploiting the spatially patterned activation of a reversibly photoswitchable fluorescent protein to reach 45- to 62-nm resolution at subsecond acquisition.3 Consequently, they were able to acquire substantially more frames at an improved signal-to-noise ratio by this techniques.
The improvement in spatio-temporal resolution achieved by PA NL-SIM is of great benefit to such studies, but this imaging modality is not currently available commercially. One of the best commercially available systems for live cell imaging that has high temporal and enhanced spatial resolution approaching that of TIRF-SIM is the Airyscan system by Zeiss.4 The Airyscan uses laser scanning confocal imaging to achieve enhanced resolution at a framing rate of 15 frames/sec with minimum photobleaching. The Airyscan technology improves the spatial resolution and signal-to-noise ratio by exploiting a combination of the equivalent of confocal imaging with a 0.2-Airy unit pinhole setting, Wiener filter-based deconvolution, and the pixel reassignment principle.
However, as with TIRF-SIM, the Airyscan fails to achieve the level of resolution required for these studies. The ability to track the motile behavior at subsecond resolution with sufficient spatial resolution remains a stumbling block and makes it difficult to conduct the motion analysis required to determine conclusively that movement of microvilli represents an efficient search strategy. Recent studies using time-resolved lattice light-sheet (LLS) microscopy and quantum dot–enabled synaptic contact mapping microscopy have shown that microvilli moving on T cell surfaces engage in a search and detection strategy that functions to improve the efficiency of ligand detection by T cells.5 Therefore, microvilli motility appears to function to increase efficiency of ligand detection as shown by these studies.
SR fluorescence microscopy remains the method of choice for nanoscale imaging of protein dynamics in living cells. As new, enhanced resolution systems become available commercially, studies like these that require long-term imaging of living cells will benefit greatly and push this important field of research forward.

References
1Wollert T, Wait E, Gandikota M, Schwarz JM, Chew TL, Langford GM (2018). F-actin mediated membrane protrusions on excitable (INS-1) cells nucleated by Arp2/3 are motile as revealed by TIRF-SIM super resolution real time live cell imaging. Mol Biol Cell 29, 3063 (Abstract # 2762).
2Orci L, Thorens B, Ravazzola M, Lodish HF (1989). Localization of the pancreatic beta cell glucose transporter to specific plasma membrane domains. Science 245, 295–247.
3Li D, Shao L, Chen BC, Zhang X, Zhang M, Moses B, Milkie DE, Beach JR, Hammer JA, Pasham M, Kirchhausen T, Baird MA, Davidson MW, Xu P, Betzig E (2015). Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 349, 944.
4Korobchevskaya K, Lagerholm CB, Colin-York H, Fritzsche M (2017). Exploring the potential of Airyscan microscopy for live cell imaging. Photonics 4, 41
5Cai E, Marchuk K, Beemiller P, Beppler C, Rubashkin MG, Weaver VM, Gérard A, Liu TL, Chen BC, Betzig E, Bartumeus F, Krummel MF (2017). Visualizing dynamic microvillar search and stabilization during ligand detection by T cells. Science 356, 598.

About the Author
George M. Langford is Distinguished Professor at Syracuse University.

About the Author:


George M. Langford is Distinguished Professor at Syracuse University.