Freeze-Fracture Immunocytochemistry

Freeze-Fracture Cytochemistry is a method that combines freeze-fracture electron microscopy with cytochemistry. It reveals the characteristics of biological membranes by labeling membrane constituents at high resolution and extensive fields of freeze-fractured membranes with cytochemical probes. Freeze-Fracture is a method useful for studying lipid membranes and their incorporated proteins in face on view.

It is a powerful technique to study the surface topology in biological membranes that reveal the pattern of integral membrane proteins. It also provides planar views of the internal organization of membranes, cells surface structure and components. Freeze - Fracture and related images have intensely enhanced the understanding of the functional morphology of the cell. This technique also reveals the presence of intra-membrane particle arrays at certain regions of plasma membrane or at contact between cells.

Freeze-Fracture Immunocytochemistry was first developed as the fracture-label technique for topology of carbohydrates in biological membranes. Cells were broken first and then labeled with immunogold probes. This method was followed by the development of the label-fracture technique, in which cell surfaces were immune-cytochemically labeled and subsequently freeze-fractured. Later, a new Freeze-Fracture cytochemistry technique has been developed combining Immunocytochemistry and Enzyme Cytochemistry which reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers like immunogold particles and cerium phosphate reaction product. This technique is an advanced method for studying the ultra-structural organization of biomembranes.

The most successful Freeze-Fracture Cytochemistry technique identified recently is Freeze-Fracture Replica Immunogold Labeling (FRIL). In FRIL, samples are first frozen fractured and replicated with platinum-carbon as in standard procedure. It is then carefully washed with Sodium Dodecyl Sulphate (SDS) to take away all the biological materials except a layer of molecules attached to the replica itself. Then Immunogold labeling of these molecules is carried out to view their membrane structure under high resolution.

The Freeze-Fracture technique consists of physical breaking apart of a frozen biological sample. The structural information available by the fracture plane is then visualized by vacuum-deposition of platinumcarbon for a replica to examine in the Transmission Electron Microscope.

The four key steps in making a freeze-fracture replica are
Rapid Freezing,
Replica Cleaning.

Freeze fracture is unique among electron microscopic techniques in providing planar views of the internal organization of membranes. Deep etching of ultra rapidly frozen samples permits visualization of the surface structure of cells and their components. Images provided by freeze fracture and related techniques have profoundly shaped our understanding of the functional morphology of the cell.

1. Cells are quickly frozen in liquid nitrogen (196C), immobilizing cell components.
2. Block of immobilized frozen cells are fractured which is irregular and occurs along lines of weakness like the plasma membrane or surfaces of organelles.
3. Surface ice is detached by a vacuum which is known as freeze etching.
4. A thin layer of carbon is evaporated vertically onto the surface to create a carbon replica.
5. Surface is concealed with a platinum vapor.
6. Organic material is digested by acid, leaving a replica.
7. Carbon-metal replica is placed on a grid and examined by a Transmission Electron Microscope.

Freeze-Fracture Cytochemistry applications include understanding of lipid droplet biogenesis, cell-surface attachment (CSAT) antigen and also new findings on PAT-family proteins. This technique contributes largely on the location of proteins within the phospholipid bilayer. The Freeze-Fracture technique is also developed to study solid-liquid dispersions. In this, a sample of dispersion is frozen, fractured and then a surface replica is made to observe under Scanning Electron Microscope. The application of the Freeze Fracture technique is also illustrated by a study of skeletal muscle plasma membrane in Duchenne muscular dystrophy.

The ultrastructure and immunocytochemistry of gap junctions in heart, brain, and spinal cord of adult rats is examined using conventional thin sections, negative staining, grid-mapped freeze-fracture replicas, and immunogold-labeled freeze-fracture replicas. We also show immunogold labeling of connexin43 in astrocyte and ependymocyte gap junctions and of connexin32 in oligodendrocyte gap junctions. Ultrastructural and freeze-fracture immunocytochemical methods have provided for definitive determination of the number, size, histological distribution, and connexin composition of gap junctions between neurons in all regions of the central nervous systems of vertebrate species (Rash, Yasumurra and Dudek). Lipid droplets are the intracellular organelles that store lipids in all organisms and are also called as Lipid Bodies or Adiposomes.

Lipid droplet biogenesis is the process of lipid droplet formation which involves fusing of newly synthesized neutral lipids in the interior of the endoplasmic reticulum membrane and subsequent budding into the cytosol. The metabolic functions of lipid droplets have been explored by studies on the lipid droplet-associated proteins, particularly the PAT family proteins, perilipin, adipophilin and TIP47. Many methods have been developed to study the nature of endoplasmic reticulum-lipid-droplet association sites for better understanding of lipid droplet formation and growth mechanisms. One of the successful methods is Freeze-Fracture Immunocytochemistry that is applied to visualize three-dimensional aspects of intracellular membranes and lipid droplets. It has the spatial resolution that discriminates closely associated membranes and organelles from one another and defines the precise location of proteins within them. The three-dimensional view provided by freeze-fracture electron microscopy reveals that at sites of close association, the lipid droplet is not situated within the endoplasmic reticulum membrane rather, both the membrane lies external to and encloses the lipid droplet. This shows that lipid droplets develop beside but not within endoplasmic reticulum membrane. Freeze-Fracture Cytochemistry shows that the PAT family proteins, adipophilin are integral components of the plasma membrane and are concentrated in the cytoplasmic leaflet of the ER membrane closely to the lipid droplet envelope.

We used freeze-fracture immunocytochemistry to localize adipophilin and TIP47, lipid droplet-associated proteins, and butyrophilin and xanthine oxidoreductase, proteins specifically involved in milk fat globule secretion, in envelopes of milk secretory granules and milk fat globules. Milk fat globule envelopes contain lipid droplet-associated proteins, such as adipophilin, TIP47, butyrophilin and xanthine oxidoreductase. The milk fat globule secretion in mammary epithelial cells presumably involves a mechanism of forming complexes between plasma membrane butyrophilin and cytosolic xanthine oxidoreductase. By using Freeze-Fracture Immunocytochemistry, locations of adipophilin, TIP47, butyrophilin, and xanthine oxidoreductase in milk fat globules and mammary epithelial cells have been identified. This technique uncovered large, unperturbed, planar expanses of biological membranes at high resolution and, combined with Immunocytochemistry has allowed to study the membrane-bound proteins specifically.

Current hypotheses of lipid droplet biogenesis, however, propose that lipids initially accumulate between the endoplasmic leaflet (E-face) and the cytoplasmic leaflet (P-face) of ER membranes (2, 22-24, 29). As the lipid pool enlarges, it pinches off the ER, generating a droplet enveloped in a phospholipid monolayer derived from the cytoplasmic leaflet of the ER membrane. Lipid droplets are amazingly heterogeneous with respect to complements and distributions of lipid droplet-associated proteins. The locations of perilipin, caveolin-1, adipophilin and TIP47 in lipid droplets of adipocytes and macrophages have been studied using freeze-fracture immunocytochemistry and electron microscopy. These lipid droplet-associated proteins are not only limited to droplet surface but pass through the lipid droplet core like caveolin-1 which is located not only in the outer membrane monolayer surrounding the lipid droplet but also occurs throughout the core of the droplet. Visualization of lipid droplet-associated proteins in lipid droplets by Freeze-Fracture Immunocytochemistry is naturally more consistent because fixation, lipid solvents, and permeabilization are avoided when compared to conventional immunofluorescence microscopic and cryosectioning methods.

Our findings from freeze-fracture immunogold labeling demonstrate unequivocally that the PAT family proteins and caveolin- 1 are not confined to the droplet envelope as maintained previously (8, 2931) and as cryosections might suggest, but clearly pervade the droplet core.

Robenek Horst is a researcher based in University of Munster, Germany and has published an array of papers in Cell Biology. Three of his important papers have been taken as reference and a study on the imaging technique - Freeze-Fracture Cytochemistry has been made in this research paper. Freeze-Fracture Cytochemistry is an important imaging technique used in the bio-research space (especially in the tissue and cellular biology) due to its ease of implementation and ability to ratify minute details. Various alternatives related to this imaging methodology have been considered for the purpose of understanding. The paper explains the Freeze-Fracture Cytochemistry in detail and aims to provide the reader an in-depth understanding of the technique.


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