Actin in Dividing Cells: Contractile Ring Filaments Bind Heavy Meromyosin

In the retina of the frog and certain other animals, melanin pigment granules move in response to light so as to shield photoreceptor outer segments. The granules are contained within the cells of the pigment epithelium (PE) which lie as a continuous sheet between the neural retina and the choroid. Moderate illumination of the eye causes the melanin granules to move from a region within a PE cell body into numerous fingerlike extensions of the cell which interdigitate with the receptor outer segments. This migration takes many minutes and is reversed when the light falling on the eye increases in intensity. Several reviews are concerned with the early descriptions of this phenomenon (6,30) and with more recent experiments (1,5,19). The mechanism of the pigment granule motion is undetermined although there are studies concerning PE ultrastructure (8, 23, 31), scanning electron microscopy of the fingerlike extensions of the PE cells (27), the role of the PE in photoreceptor phagocytosis (32), the nature of the pigment granules (19), and the action spectrum of the light which induces the migration (16). This study reports the presence of a system of microfilaments associated with the pigment granules in the fingerlike extensions processes of the PE cells. We demonstrate by heavy meromyosin (HMM) labeling that the filaments are actinlike in character and suggest that these filaments could be responsible for the migration of the melanin pigment granules.

Section: Methodsmentioning

confidence: 99%

The occurrence of actinlike filaments in association with migrating pigment granules in frog retinal pigment epithelium

Murray¹,

Dubin²

1975

“…Previous reports of bright fluorescent-HMM staining of the cleavage furrow of similar cultured cells (20) may be the result of selective actin extraction from the adjacent cytoplasm during glycerination. There is no question that a circumferential band of actin filaments, the contractile ring, is concentrated in the cleavage furrow (22,20). It seems possible that cortical actin, adjacent to the contractile ring, has gone unnoticed by electron microscopy because it is poorly preserved or less well organized than the contractile ring actin filaments.…”

mentioning

confidence: 99%

Comparison of purified anti-actin and fluorescent-heavy meromyosin staining patterns in dividing cells.

Herman¹,

Pollard²

1979

112

We purified actin antibodies by affinity chromatography from the serum of rabbits immunized with glutaraldehyde-fixed chicken gizzard actin filaments and used this anti-actin to localize actin in myofibrils and fixed cultured cells at each stage of the cell cycle. By double immunodiffusion the anti-actin reacted with both smooth and skeletal muscle actin. Several blocking and absorption experiments demonstrated that the antibodies also bound specifically to actin in nonmuscle cells. The same structures stained using either the direct or the indirect fluorescent antibody technique; and, while the indirect method was more sensitive, the direct method was superior because there was no detectable nonspecific staining. As expected, anti-actin stained the I-band of myofibrils. It also stained stress fibers and membrane ruffles in HeLa cells. Some PtK-2 cells have straight stress fibers which stained with anti-actin, but in confluent cultures all PtK-2 ceils have, instead, sinuous phase-dense fibers which stained with antibody. At prophase the whole cytoplasm stained uniformly with anti-actin. During metaphase and anaphase, anti-actin staining was concentrated diffusely in the mitotic spindle. In contrast, fluorescent heavy meromyosin stained discrete fine spindle fibers in these fixed cells. During cytokinesis, anti-actin stained the whole cytoplasm uniformly and was not concentrated in the cleavage furrow.

“…These preparative methods gave no information concerning the localization of the actin within the egg, but there is good evidence lor its presence in the cleavage furrow during cytokinesis, where it was seen first in the electron microscope as bundles of microfilaments (28,30,37). Actin has been localized in the cleavage furrow in a variety of other cell types ( 1,21,27,29,34) and this actin has been specifically labeled with heavy meromyosin (31).…”

mentioning

confidence: 99%

Preparation and purification of polymerized actin from sea urchin egg extracts.

Kane¹

1975

219

145

Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35 40~ Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KC1 before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M J~C1 and these solutions contain F-actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when l mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. The supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0~ and no heating is required.Following the demonstration of an actin-like protein, similar in properties to muscle actin, in myxomycete plasmodium (9, 10), similar methods revealed the presence of a cytoplasmic actin in unfertilized sea urchin eggs. This sea urchin egg actin was first identified and separated through its combination with rabbit muscle myosin (8,18,19), and the protein was later prepared directly from egg extracts by salting out with ammonium sulfate (17). These preparative methods gave no information concerning the localization of the actin within the egg, but there is good evidence lor its presence in the cleavage furrow during cytokinesis, where it was seen first in the electron microscope as bundles of microfilaments (28,30,37). Actin has been localized in the cleavage furrow in a variety of other cell types ( 1,21,27,29,34) and this actin has been specifically labeled with heavy meromyosin (31). The investigations reported here were begun with the aim of determining whether the methods developed for the polymerization of tubulin to microtubules in mammalian brain (2, 39) could be used to prepare tubulin from the sea urchin egg. Marine eggs are an excellent source of isolated mitotic apparatuses for the investigation of the role of microtubules in chromosome movement, but up to this time only the interaction of mammalian brain tuhulin with such isolated mitotic apparatuses has been reported (12, 25). Our attempts to prepare tubulin from extracts of sea urchin eggs with the methods developed for mammalian material were unsuccessful, and a number of modifications of this method were made to adapt it to the