Cap formation
Encyclopedia
When molecules on the surface of a cell are crosslinked, they are moved to one end of the cell to form a “cap”. This phenomenon, the process of which is called cap formation, was discovered in 1971 on lymphocytes and is a property of amoebae and all locomotory animal cells except sperm. The crosslinking is most easily achieved using a polyvalent
antibody
to a surface antigen
on the cell. Cap formation can be visualised by attaching a fluorophore
, such as fluorescein
, to the antibody.
Capping only occurs on motile cells and is therefore believed to reflect an intrinsic property of how cells move. It is an energy dependent process and in lymphocytes is partially inhibited by cytochalasin B
(which disrupts microfilaments) but unaffected by colchicine
(which disrupts microtubules). However, a combination of these drugs eliminates capping. A key feature of capping is that only those molecules that are crosslinked cap: Others do not.
Cap formation is now seen as closely related to the carbon particle experiments of Abercrombie
. In this case, crawling fibroblasts were held in a medium containing small (~1 micrometre in size) carbon particles. On occasion, these particles attached to the front leading edge of these cells: When they did so, the particles were observed to move rearward on the cell’s dorsal surface. They did so in a roughly straight line, with the particle remaining initially stationary with respect to the substratum. The cell seemed to ooze forward under the particle. In view of what we know of capping, this phenomenon is now interpreted as follows: The particle is presumably stuck to many surface molecules, crosslinking them and forming a patch. As in capping, the particle moves toward the back of the cell.
at the front of the cell and endocytosis
elsewhere has been modified by Bretscher
. He and Hopkins showed that the specific membrane endocytosed
by coated pits on motile cells is returned by exocytosis
to the cell surface at the leading edge. The spatial difference between the sites of exocytosis (at the front) and endocytosis (everywhere on the surface) leads to a flow of the matrix of the plasma membrane — lipids — from the front toward the rear. Large objects, such as patches, would be swept along with this flow, whereas non-crosslinked small molecules would be able to diffuse by Brownian motion
against the flow and so evade being swept backward. Hence, in this theory, the need for crosslinking. Bretscher proposed that on stationary cells exocytosis is random — and therefore a major difference between motile and nonmotile cells.
cytoskeleton. The molecular mechanism for how this could be achieved is unclear, since, when glycolipids or GPI-bound proteins (in the outer monolayer of the cell’s surface bilayer) are crosslinked, they cap, just like any surface protein. As these molecules cannot themselves interact directly with the cytoplasmic actin cytoskeleton, this scheme seems unlikely.
s that often act as the feet of the cell to attach it to the substrate. The the force required to rake the surface could be provided by the actin cytoskeleton.
Polyvalent
In chemistry, polyvalence or multivalence refers to species that are not restricted to a distinct number of valence bonds....
antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
to a surface antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
on the cell. Cap formation can be visualised by attaching a fluorophore
Fluorophore
A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
, such as fluorescein
Fluorescein
Fluorescein is a synthetic organic compound available as a dark orange/red powder soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications....
, to the antibody.
Steps in cap formation
- The antibody is bound to the cell. If the antibody is non-crosslinking (such as a FabFragment antigen bindingThe fragment antigen-binding is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain. These domains shape the paratope — the antigen-binding site — at the amino terminal end of the monomer...
antibody fragment), the bound antibody is uniformly distributed. This can be done at 0 °C, room temperature, or 37 °C. - If the antibody is crosslinking and bound to the cells at 0 °C, the distribution of antibodies has a patchy appearance. These “patches” are two-dimensional precipitates of antigen-antibody complex and are quite analogous to the three-dimensional precipitates that form in solution.
- If cells with patches are warmed up, the patches move to one end of the cell to form a cap. In lymphocytes, this capping process takes about 5 minutes. If carried out on cells attached to a substratum, the cap forms at the rear of the moving cell.
Capping only occurs on motile cells and is therefore believed to reflect an intrinsic property of how cells move. It is an energy dependent process and in lymphocytes is partially inhibited by cytochalasin B
Cytochalasin B
Cytochalasin B is a cell-permeable mycotoxin. It inhibits cytoplasmic division by blocking the formation of contractile microfilaments. It inhibits cell movement and induces nuclear extrusion. Cytochalasin B shortens actin filaments by blocking monomer addition at the fast-growing end of polymers....
(which disrupts microfilaments) but unaffected by colchicine
Colchicine
Colchicine is a medication used for gout. It is a toxic natural product and secondary metabolite, originally extracted from plants of the genus Colchicum...
(which disrupts microtubules). However, a combination of these drugs eliminates capping. A key feature of capping is that only those molecules that are crosslinked cap: Others do not.
Cap formation is now seen as closely related to the carbon particle experiments of Abercrombie
Michael Abercrombie
Michael Abercrombie FRS was a British cell biologist and embryologist.He was the son of the poet Lascelles Abercrombie.-External links:* http://www.ijdb.ehu.es/web/paper.php?doi=10761842...
. In this case, crawling fibroblasts were held in a medium containing small (~1 micrometre in size) carbon particles. On occasion, these particles attached to the front leading edge of these cells: When they did so, the particles were observed to move rearward on the cell’s dorsal surface. They did so in a roughly straight line, with the particle remaining initially stationary with respect to the substratum. The cell seemed to ooze forward under the particle. In view of what we know of capping, this phenomenon is now interpreted as follows: The particle is presumably stuck to many surface molecules, crosslinking them and forming a patch. As in capping, the particle moves toward the back of the cell.
"Flow"
Abercrombie thought that the carbon particle is a marker on the cell surface and its behaviour reflects what the surface is doing. This led him to propose that, as a cell moves, membrane from internal stores is added at the front of the cell — enabling the cell to extend forward — and retrieved toward the rear of the cell. This process of exocytosisExocytosis
Exocytosis , also known as 'The peni-cytosis', is the durable process by which a cell directs the contents of secretory vesicles out of the cell membrane...
at the front of the cell and endocytosis
Endocytosis
Endocytosis is a process by which cells absorb molecules by engulfing them. It is used by all cells of the body because most substances important to them are large polar molecules that cannot pass through the hydrophobic plasma or cell membrane...
elsewhere has been modified by Bretscher
Mark Bretscher
Mark Bretscher is a British biological scientist and Fellow of the Royal Society. He works at the Medical Research Council Laboratory of Molecular Biology in Cambridge, United Kingdom...
. He and Hopkins showed that the specific membrane endocytosed
Endocytosis
Endocytosis is a process by which cells absorb molecules by engulfing them. It is used by all cells of the body because most substances important to them are large polar molecules that cannot pass through the hydrophobic plasma or cell membrane...
by coated pits on motile cells is returned by exocytosis
Exocytosis
Exocytosis , also known as 'The peni-cytosis', is the durable process by which a cell directs the contents of secretory vesicles out of the cell membrane...
to the cell surface at the leading edge. The spatial difference between the sites of exocytosis (at the front) and endocytosis (everywhere on the surface) leads to a flow of the matrix of the plasma membrane — lipids — from the front toward the rear. Large objects, such as patches, would be swept along with this flow, whereas non-crosslinked small molecules would be able to diffuse by Brownian motion
Brownian motion
Brownian motion or pedesis is the presumably random drifting of particles suspended in a fluid or the mathematical model used to describe such random movements, which is often called a particle theory.The mathematical model of Brownian motion has several real-world applications...
against the flow and so evade being swept backward. Hence, in this theory, the need for crosslinking. Bretscher proposed that on stationary cells exocytosis is random — and therefore a major difference between motile and nonmotile cells.
"Cytoskeleton"
An alternative view is that the patches are moved to the rear of the cell by direct attachment to the actinActin
Actin is a globular, roughly 42-kDa moonlighting protein found in all eukaryotic cells where it may be present at concentrations of over 100 μM. It is also one of the most highly-conserved proteins, differing by no more than 20% in species as diverse as algae and humans...
cytoskeleton. The molecular mechanism for how this could be achieved is unclear, since, when glycolipids or GPI-bound proteins (in the outer monolayer of the cell’s surface bilayer) are crosslinked, they cap, just like any surface protein. As these molecules cannot themselves interact directly with the cytoplasmic actin cytoskeleton, this scheme seems unlikely.
"Rake"
A third scheme, by de Petris, suggests that a motile cell is continuously raking its surface from front to back: Any aggregates (but not uncrosslinked molecles) caught in the teeth of the rake are moved to the back of the cell. In this scheme, the nature of the tines of the rake are not specified but could, for example, be surface integrinIntegrin
Integrins are receptors that mediate attachment between a cell and the tissues surrounding it, which may be other cells or the ECM. They also play a role in cell signaling and thereby regulate cellular shape, motility, and the cell cycle....
s that often act as the feet of the cell to attach it to the substrate. The the force required to rake the surface could be provided by the actin cytoskeleton.