A new approach for the direct visualization of the membrane cytoskeleton in cryo-electron microscopy: a comparative study with freeze-etching electron microscopy

Abstract

An improved unroofing method consisting of tearing off the cell membrane using an adhesive electron microscopy (EM) grid instead of vitreous ice sectioning (cryo-sectioning) has enabled us to panoramically view the membrane cytoskeleton in its native state with extremely high contrast. Grids pre-treated with Alcian blue were placed on cells, and a portion of the dorsal plasma membrane was transferred onto the grid, which was then floated in buffer solution. These membrane fragments contained sufficient cytoskeleton and were of suitable thickness for observation by cryo-EM. Many actin filaments and microtubules were clearly observed on the cytoplasmic surface of the plasma membrane with extremely high contrast because the soluble components of the cytoplasm flowed out and broke away from the cells. Actin filaments extended in all directions in a smooth contour with little branching. Microtubules spread out as far as 3 µm or more while winding gently in their native state. Upon fixation with 1% glutaraldehyde, however, the microtubules became straight and fragmented. Cryo-EM revealed for the first time a smooth endoplasmic reticulum network beneath the cell membrane in native cells. Clathrin coats and caveolae were also observed on the cytoplasmic surface of the plasma membrane, similar to those seen using freeze-etching replica EM (freeze-etching EM). Unroofing was also useful for immuno-labelling in cryo-EM. Antibody-labelled IQGAP1, one of the effector proteins facilitating the formation of actin filament networks, was localized alongside actin filaments. Freeze-etching EM confirmed the morphological findings of cryo-EM.

Rapid, directed transport of DC-SIGN clusters in the plasma membrane

“…Imaging of DC-SIGN clusters in cells expressing a fluorescent construct containing the MT-binding domain of ensconsin demonstrated that many cluster trajectories are coincident with MTs. Recent electron microscopy studies demonstrate that some MTs are present in close proximity to the plasma membrane ( 45 ). Moreover, the MT-depolymerizing drug nocodazole, but not latrunculin A, which disrupts F-actin, suppresses a fraction of clusters undergoing rapid, directed movement, further implicating MTs in this phenomenon.…”

The Evolving Complexity of the Podocyte Cytoskeleton

“…46 Recent in vitro studies advanced our understanding of the architecture of the actin cytoskeleton. 47,48 However, advances in new super resolution microscopy techniques such as stimulated emission depletion or stochastic optical reconstruction microscopy now offer new avenues to fully understand the complexity, composition, and dynamics of the podocyte actin cytoskeleton in vivo. 49 Additional insights into the structural and functional relation of FPs have come to light through the study of dramatic changes in podocyte morphology which take place during glomerular development, where cuboidal nonfiltering podocytes differentiate into arborized octopus-like filtering cells 50 (Figure 1).…”

Scattered podosomes and podosomes associated with the sealing zone architecture in cultured osteoclasts revealed by cell shearing, quick freezing, and platinum‐replica electron microscopy

“…Although the presently employed methods failed to reveal the total network of ER throughout the cell, they did provide structural evidence of the relationship between podosomes and smooth ER in the OC. Some types of the smooth ER network can be visualized reliably only by using quick freezing techniques (Kanaseki et al, 1998;Makihara et al, 2016). Previous studies by replica electron microscopy pointed out the presence of variable types of membrane components termed variously as ER (Aggeler, Takemura, & Werb, 1983;Makihara et al, 2016), membranous organelle (Isobe, Warner, & Lemanski, 1988), anastomotic membrane tubule (Heuser & Anderson, 1989), and putative membrane component (Svitkina, Verkhovsky, & Borisy, 1996).…”