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Retraction Fibers

Introduction
In 1963, while using light microscopy and TEM, researchers Taylor and Robbins uncovered what they recorded to be long, tube-like structure present during cell migration. They named these structures retraction fibrils and they were later named retraction fibers.

Retraction fibers differ from filopodia in that they stem from retraction, hence their name, as opposed to protrusion. Containing no microtubules, the main component of retraction fibers are thin microfilaments of actin that form fibrous bundles giving them their fiber structure. As actin-rich cytoplasmic extensions, retraction fibers can be observed at the trailing edge of cells that are migrating forward while attached to a substrate. During cell migration, retraction fibers are left in a network behind the migrating cell. They elongate as the cell migrates along the substrate until they break.

Retraction fibers are first detectable during early mitosis but they have been shown to persist throughout the post-mitotic stage. This finding correlates to their functional capacity to orient the mitotic spindle which they do using external, cortical cues. In order to orient the spindle, retraction fibers must associate with the plasma membrane, cytoskeletal proteins, and adhesion factors.

Retraction fibers have also been found to have their own associated structure called the migrasome. The elongating retraction fiber forms vesicles at its tips and ends which bud off, releasing the migrasome which then releases the cytoplasmic contents it has absorbed in order for them to be released into the extracellular matrix or taken up by other cells via endocytosis.

Migrasome
A migrating cell will form a trail of retraction fibers behind it leaving vesicles known as migrasomes budding at the tips of the trailing retraction fibers. The cytosolic contents of a cell are transported into migrasomes and subsequently released into the extracellular space or taken up by a neighboring cell via endocytosis.

When observed under TEM, migrasomes are oval-shaped, membrane-bound vesicular structures present in the extracellular space around a cell. The distance between the migrasome and the periphery of the cell varies but migrasomes tend to cluster on one pole's edge. They contain many other smaller vesicles in their lumen ranging from 10 to 300 in quantity. Visually, migrasomes have been described to look like open pomegranates.

Migrasomes follow a relatively predictable growth pattern. The first phase is a period of rapid growth during which their maximum size is reached, between 3µm - 10µm in diameter, and the attached retraction fibers are structurally stable. The second phase is characterized by a plateau in growth, and the size of the migrasome remains relatively unchanged. In the final stage, following the disassembly of the retraction fiber, the migrasome is released into the extracellular space. The growth cycle begins approximately 40 min after the cell begins migration, at this point in time migrasomes begin to bud on the trailing retraction fibers.

The initiation of the migrasome growth cycle is dependent upon migration of the cell and retraction fibers, since migrasomes grow on retraction fibers and retraction fibers are localized to the migration path of the cell.

Actin
Retraction fibers are essentially tubules that are comprised of actin in the form of fibrous bundles. The polymerization of the actin in these bundles is the mechanism by which retraction fibers are able to promote or inhibit cell migration. As spreading occurs of a cell, ventral actin filaments in the retraction fibers remain stationary in the edge as they are immobilized by adhesion factors. Dorsal actin filaments are not immobilized and move along the edge relative to the substrate, the migrating cell.

ERM protein family
The closely related family of actin-binding proteins including ezrin, radixin, and moesin have all been found to be localized to retraction fibers. Specifically, the three ERM proteins localize themselves in the ends of the retraction fibers near the cell cortex. In this location, the ERM protein family contributes to the role of retraction fibers in organization of the mitotic spindle by constituting cortical cues. Structurally, ezrin, radixin, and moesin function to crosslink the actin filaments of the fibers to the plasma membrane via their FERM domain.

Ezrin
Retraction fibers are highly enriched in the protein ezrin, found in actin-containing surfaces, as they are primarily composed of actin filaments. Its function, as with the other ERM proteins, is to link the plasma membrane with components of the cytoskeleton.

Radixin
Radixin, part of the ERM protein family, is localized in retraction fibers where it plays an important role in joining actin filaments to adhesion molecules in the plasma membrane. The role of radixin in general, like the other ERM proteins, is crucial to the organization and structure of the cortical cytoskeleton during migration.

Moesin
The expression of moesin in varies across cell types more than its paralog ezrin but its distribution is not random, specifically in the cortical skeleton, as it is here where it is localized in retraction fibers. Distribution of moesin follows changes that occur in the cells during movement which relates to the function of retraction fibers being to restructure both the plasma membrane and the membrane skeleton.

Mechanism
The function of the retraction fibers is to orient the mitotic spindle. It has been proposed that factors causing polarization are recruited to the cell cortex by retraction fibers, which leads to this orientation. Through the use of ablation experiments, the orientation of the mitotic spindle by external cues from the retraction fibers has been documented.

Retraction fiber ablation also leads to cell deformation, indicating that they exert a pulling force on the cell body. These forces are key polarizing signals during mitosis, which are similar to the forces during interphase that trigger mechanotransduction - the process by which the cell converts mechanical stimulus into electrochemical activity. From the base to the cell body, a HeLa cell's retraction fibers have average 13±3 μm in length with a tension of 245±42 pN. These forces are the cause of the deformation of cells. Actin filament assembly inhibition or the inhibition of myosin motor activity leads to an elongation of the cell body in the axis of the retraction fibers (when the retraction fibers have been placed in vitro to be acting on certain parts and axes of the cell). Treating cells with calyculin A, which increases myosin activity, results in the rounding of the cell body. It is therefore inferred that the mitotic cell shape is dependent on the contractile forces internal to the cell that act on the external forces of the retraction fiber that oppose them. When retraction fibers were ablated on a certain axis, there was a disturbance in the force balance, which lead to contractile behaviour of the cells in the axis of the fibers that had been ablated. Changing the position and distribution of retraction fibers while the cell is in the process of rounding results in specific forces being exerted on the body of the cell. This leads to a polarized contraction of the cell. Therefore, while a cell is rounding, it tends to pull stronger in the axis where more retraction fibers are present. The retraction fiber forces exerted on mitotic cells induce spindle rotation. These forces are able to influence subcortical actin structures that interact with astral microtubules, leading to mitotic spindle alignment with the external forces. Studies on mitotic Dictyostelium discoideum cells show that when forces are exerted on them, they initiate myosin II and cortexillin, which is involved in the cross-linking of actin. Also, studies on mitotic Ptk2 cells show an agreggation of myosin II and actin at the foot of retraction fibers leading to the assumption that forces exerted by retraction fibers may cause the formation of acto-myosin complexes, leading to cell polarization and spindle orientation. Polarization of the subcortical actin formations cannot be induced by the spindle alone and the amount and depth of integration of subcortical actin structures into a cell's cytoplasm are dependent on the relative distribution of retraction fibers. After an ablation experimented was done on retraction fibers facing each pole of the spindle, actin structures repolarized along the retraction fibers that remained. To further demonstrate that actin structure repolarization is responsible for spindle rotation, rotation of the mitotic spindle is always followed by the repolarization of actin.