Seymour H. Wollman

Mitosis in rat thyroid epithelial cells in vivo. I. Ultrastructural changes in cytoplasmic organelles during the mitotic cycle.

The ultrastructure of mitosis was studied in polarized, well-differentiated thyroid epithelial cells in vivo following stimulation with the goitrogen, thiouracil, and attention was focused on changes occurring in cytoplasmic organelles. RER cisternae appeared from mid-prometaphase to mid-telophase to undergo a vesicular transformation from lamellar profiles to smaller, rounder profiles with an up to 60% lower concentration of bound ribosomes. The Golgi apparatus underwent a nearly parallel breakdown/reformation process, and Golgi remnants were frequently associated with large clouds of smooth-surfaced 70-nm-diameter vesicles. Apical vesicles, believed to contain thyroglobulin, were found to accumulate during mitosis, reaching about six times their interphase concentration by mid-telophase, when they were observed in large clusters about the spindle poles. Clusters of apical vesicles then appeared to migrate to their usual apical location. Changes in the distribution of lysosomes were noted in late prometaphase, when they became aggregated into clusters, and in early telophase when they accumulated near the cleavage furrow.

Mitosis in rat thyroid epithelial cells in vivo. IV. Cell surface changes.

Cell surface changes associated with mitosis in thyroid epithelial cells were studied by transmission electron microscopy. The apical surface showed a decline of about 40% in the linear concentration of microvilli observed in longitudinally sectioned cells from metaphase through most of telophase. No pseudopods were observed projecting from the apical surface of mitotic cells until late telophase, when a single pseudopod was found. The possibility of relatively increased macropinocytosis in late telophase was also compatible with the finding of an increase in the number and volume of colloid droplets at this time to three times the amount found in any other portion of the mitotic cycle. The lateral and basal cell surfaces did not change markedly during mitosis, but tended to show an increased number of interdigitations and infoldings. The furrow surface was characterized by a transient increase in microvilli on its apical half and by the frequent appearance of pleomorphic blebs on the basal side.

Embedding in a collagen gel stabilizes the polarity of epithelial cells in thyroid follicles in suspension culture

Separated thyroid follicles are stable in suspension culture in Coons modified Hams F12 medium containing 0.5% calf serum. They resemble follicles in vivo except for the absence of a basal lamina. However, the epithelial cells reverse polarity and the follicles invert when the serum concentration is raised to 5%. A number of substances, especially components of extracellular matrix, were added to the medium to ascertain if they could stabilize the follicles against inversion in 5% serum. Cellular and plasma fibronectin, gelatin, heat-denatured collagen, methylcellulose and laminin did not stabilize. The addition to the medium of as little as 50 micrograms/ml of acid-soluble collagen prepared from calf skin or rat tail tendons resulted in the formation of small clouds of gel. Follicles embedded within the gel were stabilized. Follicles in the same dish but not embedded in the gel inverted. Stabilization was not specific for collagen, since follicles embedded in a plasma clot were also stabilized. A gel was not sufficient for stabilization, since embedding in an agarose gel did not stabilize. Ultrastructural studies indicate that adherence to a limited number of gelled fibers of collagen covering only a small fraction of the basal plasma membrane may be sufficient to stabilize and that a basal lamina formed in the presence of laminin but without added collagen does not stabilize.

Mitosis in rat thyroid epithelial cells in vivo: II. Centrioles and pericentriolar material

The growth, movements, and orientations of centrioles and the behavior of pericentriolar material during mitosis in vivo was studied at the ultrastructural level in stimulated rat thyroid epithelial cells. Apparent biogenesis of daughter centrioles was first observed in interphase and by very early prophase, daughter centrioles had elongated to about two-thirds of their mature length. They appeared to retain this intermediate length throughout most of mitosis and were observed to reach their mature length only in very late telophase or interphase. Very early in mitosis, centriolar diplosomes left their interphase position near the cell apex and individual diplosomes were observed in various locations, including close to the base of the cell. However, by metaphase (and later) diplosomes were consistently found about midway between the apex and the base, corresponding to the establishment of a spindle axis parallel to the follicular luminal surface. Measurements of the distance between centriolar diplosomes during mitosis revealed a steadily increasing separation until mid-telophase.

Ultrastructure of erythrophagocytosis and red blood cell fission by thyroid epithelial cells in Vivo

Rats fed the goitrogen, thiouracil, were found to undergo a microhemorrhagic process within the thyroid gland, sometimes resulting in the deposition of red blood cells (RBCs) within follicular lumens. In such instances, erythrophagocytosis by follicular epithelial cells was frequently observed. This involved progressive RBC engulfment by an apical epithelial pseudopod, which conformed closely to the contour of the (often irregular) RBC and appeared to almost totally exclude luminal colloid from the completed phagosome. Completed phagosomes were retracted into the apical epithelial cytoplasm, and a periphagosomal accumulation of 7-nm microfilaments was often observed at this stage. Another feature of thyroid epithelial erythrophagocytosis was the occasional finding of apparent pseudopod-induced RBC fission. This was suggested by the observation of narrow, elongated RBC necks, surrounded by thickened pseudopods containing densely packed microfilments immediately adjacent to the RBC necks. It seems likely that these microfilaments play a role in the fission process, possibly functioning as a contractile ring.

Mitosis in thyroid follicular epithelial cells in vivo. III. Cytokinesis.

The ultrastructure of cytokinesis in vivo was studied in rat thyroid follicular epithelial cells. Where it could be ascertained, the spindle axis appeared tangential and the cleavage furrow perpendicular to the follicular luminal surface. The furrowing process was associated with a typical microfilamentous contractile ring and resulted in the formation of a short intercellular bridge (IB), usually located initially about midway between the base and apex of the daughter cells. The IB then appeared to undergo a migration process and was observed progressively closer to the apical surface. Just basal to the apically migrated IB, a new tight junction was observed between the daughter cells and—in presumed continuity with nearby retained tight junctions to neighboring cells—eventually formed a seal basal to the IB prior to its removal. Final separation of the daughter cells occurred without apparent stretching of the IB. The IB underwent degenerative changes and eventually appeared to be phagocytosed by one of the epithelial cells.

Ultrastructure and some other properties of inverted thyroid follicles in suspension culture.

Abstract Separated thyroid follicles in suspension culture invert in 5% serum. In some, the inversion is not complete in that a small normal follicle persists completely in the interior of an inverted follicle. In inverted follicles the lumens are distended and electron lucent. The bounding epithelial cells are stretched, have relatively few microvilli on the surface toward the medium but they have bundles of oriented microfilaments usually located near the lumen. The cells are connected together by tight junctions. When inverted follicles are punctured, the lumen shrinks, the cells retract and become cuboidal and microvilli reappear. Microfilaments persist at the luminal surface but no longer in oriented bundles. No appreciable extracellular matrix is present at the basal cell surface in contact with the lumen, but matrix is occasionally observed between cells. Since bundles of microfilaments like stress fibers are observed in the cells in suspension culture, the presence of stress fibers in cells in monolayer culture is probably not dependent on attachment but might be a reflection of the spreading of the attached cells.

Ultrastructural aspects of capillary fusion during the development of thyroid hyperplasia

When young adult male Fischer rats are fed a low-iodine diet containing 0.25% thiouracil the blood capillaries enlarge and appear to fuse, with the temporary appearance of partial septa between capillary lumina. The mechanism of the fusion was examined by electron microscopy. The partial septum was made up of intact cells with a tight junction near its distal end. A mechanism to form such partial septa must involve the penetration, by endothelial cells, of two basal laminae and a collagen layer between the capillary walls. The penetration may be made by pseudopods on the abluminal surface of many endothelial cells, about half of which penetrated basal laminas. The fusion may involve pairs of pseudopods at cell margins. Multiple small fusion sites between neighboring walls of parallel capillaries observed in serial sections may be an intermediate stage in capillary fusion. Aggregates of blood platelets were observed near collagen exposed when endothelial cells retracted.

Change of inverted thyroid follicle into a spheroid after embedding in a collagen gel

When inverted thyroid follicles in suspension culture are embedded in a collagen gel, there is extensive reorganization of the follicle. To identify intermediate stages in the reorganization, a suspension of inverted follicles was mixed with a cold solution of collagen (0.1 mg/ml) in culture medium and the resultant was warmed and allowed to gel. Prior to embedding, the epithelial cells bounding the lumens formed a monolayer of attenuated cells with their microvilli-bearing surface in contact with the medium. The first change noted was a shrinkage of the lumen in many follicles by 18 h. The cells became cuboidal to columnar. Some of the cells had long sheet-like processes extending into the lumen in contact with those of other cells. In late stages of the reorganization, 48 h, the cells were arranged in a compact spheroid. The spheroids contained two different kinds of colloid-filled lumens, possibly of different origins, one a spherical microlumen, the other very long and narrow in section. The peripheral cells of the spheroid had a smooth plasma membrane (without microvilli) in contact with collagen. Although most of the cells in a section had a microvilli-bearing surface forming part of the boundary of a lumen, it is not certain that all cells were in contact with a lumen.

Pseudopod behavior in hyperplastic thyroid follicles in vivo

As an early step in the secretion of thyroid hormones, colloid droplets are formed by pseudopods of thyroid epithelial cells. However, such droplets are generally absent from thyroid stimulating hormone (TSH)-stimulated thyroid follicles in places where luminal colloid has been largely resorbed. To investigate the reason for this absence, stimulated thyroid follicles were examined. Even in markedly colloid-depleted lumens, pseudopods were frequently observed. These were often short and flat, abutting against the opposing epithelial surface and following its contour. No room appeared available for these pseudopods to roll up to form colloid droplets. In addition, ultrastructural changes were often visible in the opposing surface just subjacent to regions of pseudopod contact. These changes consisted of the loss of apical microvilli and the formation of an organelle-excluding microfilamentous meshwork, suggestive of early psuedopod formation. The possibility that contact with a pseudopod might be capable of inducing the formation of a second pseudopod was also suggested by the frequent finding of pairs of pseudopods, one abutting against or partially enveloping another.