Structural change in the cell center during fibroblast polarization and movement

In polarizing and migrating 3T3/Balb mouse fibroblasts, the centrioles are located between the nucleus and the leading edge of the cell. In cells within the monolayer and in migrating cells, the centrioles have a random orientation towards the substrate. In polarized cells, that still remain in the monolayer, one centriole may be perpendicular to the substrate plane in 70% of cases. Upon polarization and migration of fibroblasts, the number of microtubules, which radiate from the centriolar region, increases. These data support a hypothesis that the number of microtubules in the cell centre characterizes the rate of their renovation in the cytoplasm. It is concluded that the cell centre is strongly involved in polarization and migration of fibroblasts.     

Behavior of the cell center during epithelial cell spreading

In the epithelial cells of mouse embryo renal channels, centrioles are located near the plasma membrane of the apical part of the cell. In most of the cells an active centriole carries a cilium, which comes out into the channel lumen. In the epithelial cells, suspended after trypsinisation and in single cells adhering to the substrate, the centrioles are located near the nucleus, and the outcoming cilia are not observed. In the spread cells of epithelial islets, the centrioles are also found near the nucleus, and in most cases an active centriole carries a cilium, which comes out of the cytoplasm at the upper side of the cell. In the peripheral cells of the islet, centrioles are positioned between the nucleus and the active edge of the cell. In the epithelial cells in situ, a relatively small number of microtubules radiate from the active centrioles. In the suspended cells, the activation of microtubule formation is observed in the cell center. In the spread cells of the epithelial islets there occurs a further increase in the number of microtubules radiating from the active centrioles. In the peripheral cells which cause translocation of the epithelial islet in the culture, the number of microtubules, radiating from the centrioles does not differ significantly from that of the inner cells of the islet. The cell center of the epithelial cells does not seem to be actively involved in the locomotion of the epithelial cells in the culture.     

Spermatogenesis and sperm structure of Acerella muscorum, (Ionescu, 1930) (Hexapoda, Protura)

The general organization of the male genital system, the spermatogenesis and the sperm structure of the proturan Acerella muscorum have been described. At the apex of testis apical huge cells are present; their cytoplasm contains a conventional centriole, a large amount of dense material and several less electron-dense masses surrounded by mitochondria. Spermatocytes have normal centrioles and are interconnected by cytoplasmic bridges. Such bridges seem to be absent between spermatid cells and justify the lack of synchronization of cell maturation. Spermatids are almost globular cells with a spheroidal nucleus and a large mass of dense material corresponding to the centriole adjunct. Within this mass a centriole is preserved. Mitochondria of normal structure are located between the nucleus and the plasma membrane. The spermatids are surrounded by a thick membrane. No flagellar structure is formed. Sperm have a compact spheroidal nucleus, a large cap of centriole adjunct material within which a centriole is still visible. A layer of mitochondria is located over the nucleus. The cytoplasm is reduced in comparison to spermatids; many dense bodies are interspersed with sperm in the testicular lumen. The sperm are small, immotile cells of about 2.5-3 μm in diameter.     

The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells

We have generated several stable cell lines expressing GFP-labeled centrin. This fusion protein becomes concentrated in the lumen of both centrioles, making them clearly visible in the living cell. Time-lapse fluorescence microscopy reveals that the centriole pair inherited after mitosis splits during or just after telophase. At this time the mother centriole remains near the cell center while the daughter migrates extensively throughout the cytoplasm. This differential behavior is not related to the presence of a nucleus because it is also observed in enucleated cells. The characteristic motions of the daughter centriole persist in the absence of microtubules (Mts). or actin, but are arrested when both Mts and actin filaments are disrupted. As the centrioles replicate at the G1/S transition the movements exhibited by the original daughter become progressively attenuated, and by the onset of mitosis its behavior is indistinguishable from that of the mother centriole. While both centrioles possess associated gamma-tubulin, and nucleate similar number of Mts in Mt repolymerization experiments. during G1 and S only the mother centriole is located at the focus of the Mt array. A model, based on differences in Mt anchoring and release by the mother and daughter centrioles, is proposed to explain these results.     

Centrioles in the cell cycle of L cells

The structure of centrosome in non-synchronous L-cells culture during the cell cycle has been studied. In mitosis, mother and daughter centrioles, which differ in their ultrastructure, are located perpendicularly in the pole of the spindle. Microtubules, meeting in the pole area terminate mainly in electron-dense clottings of fibrillar matter surrounding the diplosoma. In telophase, disjunction of mother and daughter centrioles begins. At the beginning of G1-period, centrioles move off from each other for several micron, and then draw together again without forming diplosome. Pericentriolar satellites form on mother centriole of some cells at this time, they disappear at the beginning of S-period, replication of centrioles begins; daughter centrioles reach the size of mother centrioles in anaphase. During growth and maturation, centrioles in L-cells undergo structural changes similar to those described for SPEV cells (Vorob'ev, Chentsov, 1982). Several types of meeting points for microtubules exist in L-cells during the whole interphase: surface of centrioles per se, pericentriolar satellites, free foci.     

Vertebrate primary cilia: a sensory part of centrosomal complex in tissue cells,but a “sleeping beauty” in cultured cells?

Primary cilium development along with other components of the centrosome in mammalian cells was analysed ultrastructurally and by immunofluorescent staining with anti-acetylated tubulin antibodies. We categorized two types of primary cilia, nascent cilia that are about 1microm long located inside the cytoplasm, and true primary cilia that are several microm long and protrude from the plasma membrane. The primary cilium is invariably associated with the older centriole of each diplosome, having appendages at the distal end and pericentriolar satellites with cytoplasmic microtubules emanating from them. Only one cilium per cell is formed normally through G(0), S and G(2)phases. However, in some mouse embryo fibroblasts with two mature centrioles, bicilates were seen. Primary cilia were not observed in cultured cells where the mature centriole had no satellites and appendages (Chinese hamster kidney cells, line 237, some clones of l-fibroblasts). In contrast to primary cilia, striated rootlets were found around active and non-active centrioles with the same frequency. In proliferating cultured cells, a primary cilium can be formed several hours after mitosis, in fibroblasts 2-4 h after cell division and in PK cells only during the S-phase. In interphase cells, formation of the primary cilium can be stimulated by the action of metabolic inhibitors and by reversed depolymerization of cytoplasmic microtubules with cold or colcemid treatments. In mouse renal epithelial cells in situ, the centrosome was located near the cell surface and mature centrioles in 80% of the cells had primary cilium protruding into the duct lumen. After cells were explanted and subcultured, the centrosome comes closer to the nucleus and the primary cilium was depolymerized or reduced. Later primary cilia appeared in cells that form islets on the coverslip. However, the centrosome in cultured ciliated cells was always located near the cell nucleus and primary cilium never formed a characteristic distal bulb. A sequence of the developmental stages of the primary cilium is proposed and discussed. We also conclude that functioning primary cilium does not necessarily operate in culture cells, which might explain some of the contradictory data on cell ciliation in vitro reported in the literature.     

Orientation of spindle axis and distribution of plasma membrane proteins during cell division in polarized MDCKII cells

MDCKII cells differentiate into a simple columnar epithelium when grown on a permeable support; the monolayer is polarized for transport and secretion. Individual cells within the monolayer continue to divide at a low rate without disturbing the function of the epithelium as a barrier to solutes. This presents an interesting model for the study of mitosis in a differentiated epithelium which we have investigated by confocal immunofluorescence microscopy. We monitored the distribution of microtubules, centrioles, nucleus, tight junctions, and plasma membrane proteins that are specifically targeted to the apical and basolateral domains. The stable interphase microtubule cytoskeleton was rapidly disassembled at prophase onset and reassembled at cytokinesis. As the interphase microtubules disassembled at prophase, the centrioles moved from their interphase position at the apical membrane to the nucleus and acquired the ability to organize microtubule asters. Orientation of the spindle parallel to the plane of the monolayer occurred between late prophase and metaphase and persisted through cytokinesis. The cleavage furrow formed asymmetrically perpendicular to the plane of the monolayer initiating at the basolateral side and proceeding to the apical domain. The interphase microtubule network reformed after the centrioles migrated from the spindle poles to resume their interphase apical position. Tight junctions (ZO-1), which separate the apical from the basolateral domains, remained assembled throughout all phases of mitosis. E-cadherin and a 58-kD antigen maintained their basolateral plasma membrane distributions, and a 114- kD antigen remained polarized to the apical domain. These proteins were useful for monitoring the changes in shape of the mitotic cells relative to neighboring cells, especially during telophase when the cell shape changes dramatically. We discuss the changes in centriole position during the cell cycle, mechanisms of spindle orientation, and how the maintenance of polarized plasma membrane domains through mitosis may facilitate the rapid reformation of the polarized interphase cytoplasm.     

Centrioles in the cell cycle. I. Epithelial cells

A study was made of the structure of the centrosome in the cell cycle in a nonsynchronous culture of pig kidney embryo (PE) cells. In the spindle pole of the metaphase cell there are two mutually perpendicular centrioles (mother and daughter) which differ in their ultrastructure. An electron-dense halo, which surrounds only the mother centriole and is the site where spindle microtubules converge, disappears at the end of telophase. In metaphase and anaphase, the mother centriole is situated perpendicular to the spindle axis. At the beginning of the G1 period, pericentriolar satellites are formed on the mother centriole with microtubules attached to them; the two centrioles diverge. The structures of the two centrioles differ throughout interphase; the mother centriole has appendages, the daughter does not. Replication of the centrioles occurs approximately in the middle of the S period. The structure of the procentrioles differs sharply from that of the mature centriole. Elongation of procentrioles is completed in prometaphase, and their structure undergoes a number of successive changes. In the G2 period, pericentriolar satellites disappear and some time later a fibrillar halo is formed on both mother centrioles, i.e., spindle poles begin to form. In the cells that have left the mitotic cycle (G0 period), replication of centrioles does not take place; in many cells, a cilium is formed on the mother centriole. In a small number of cells a cilium is formed in the S and G2 periods, but unlike the cilium in the G0 period it does not reach the surface of the cell. In all cases, it locates on the centriole with appendages. At the beginning of the G1 period, during the G2 period, and in nonciliated cells in the G0 period, one of the centrioles is situated perpendicular to the substrate. On the whole, it takes a mature centriole a cycle and a half to form in PE cells.     

OBSERVATIONS ON THE CENTRIOLES OF THE SEA URCHIN SPERMATOZOON

The spermatozoon of Lytechinus variegatus has two parallel centrioles. The basal body of the flagellum consists of the proximal centriole (a short cylinder of nine tubule-triplets) and its distal extension of nine tubule-doublets. The distal centriole lies near the distal end of the basal body, between the nucleus and the mitochondrion. The observations suggest that both the proximal and the distal centrioles are polarized structures, their tubule-triplets pitched in the same direction and their distal ends associated with the flagellar axoneme and with the mitochondrion, respectively. The distal centriole in different spermatozoa occupies different positions around the basal body-flagellum complex.     

Epidermis and protonephridia of a free-living platyhelminth, Mesocastrada führmanni Voltz, 1898 (Rhabdocoela, Typhloplanoida): An ultrastructural study

The ultrastructure of the epidermis and the protonephridia of the free-living rhabdocoel Mesoscastrada führmanni is described. The epidermis consists of polarized cells, the nucleus located in the basal part of the cell and the mitochondria in the apical part. The surface is entirely covered by cilia anchored in the cytoplasm by horizontal and vertical striated rootlets. Cilia of the flame bulbs also have horizontal and vertical striated rootlets. The weir apparatus of the cyrtocyte is composed of a single row of ribs connected by a thin “membrane” of extracellular material. Bundles of microtubules, located in the ribs originate in the centrioles. Epidermal cells and flame bulbs of M. führmanni closely resemble those of the other Typhloplanoida examined so far.     

Centrioles take center stage.

Centrioles are among the most beautiful and mysterious of all cell organelles. Although the ultrastructure of centrioles has been studied in great detail ever since the advent of electron microscopy, these studies raised as many questions as they answered, and for a long time both the function and mode of duplication of centrioles remained controversial. It is now clear that centrioles play an important role in cell division, although cells have backup mechanisms for dividing if centrioles are missing. The recent identification of proteins comprising the different ultrastructural features of centrioles has proven that these are not just figments of the imagination but distinct components of a large and complex protein machine. Finally, genetic and biochemical studies have begun to identify the signals that regulate centriole duplication and coordinate the centriole cycle with the cell cycle.     

Types and Life Cycles of Centrioles of Flagellates

SUMMARY. Two centrioles, an old one and a new one, are always present in the resting cell. From prophase onward two new ones and two old ones are present. Beginning with the resting stage, five types of centriole life cycles are described and compared with one another: In type 1, both centrioles are elongate; in type 2, the old one is long and the new one, which is short, elongates in prophase; in type 3, both are short, both elongate in prophase, and both, except for their anterior tips, degenerate in late telophase; in type 4, both are long but in prophase their distal ends become free of the rest of the centrioles, these ends migrate to center or posterior end of cell, where, after they produce the achromatic figure and it completes its function in nuclear division, they degenerate; in type 5, both are short and neither elongates at any stage of its life cycle.
New centrioles are produced by the anterior ends of old ones. In their first generation, centrioles produce only extranuclear organelles (flagella, parabasals, axostyles, etc.); in their second and later generations, they produce only the achromatic figure (gametogenesis in Trichonympha and reorganization in Barbulanympha and Rhynchonympha are exceptions to this rule).
The distal ends of centrioles in some types of cycles are surrounded by centrosomes; in others they are not. In one type of centriole life cycle a small central spindle is present in the resting cell in two genera; in the other types this is not the case.     

Centrosome-centriole abnormalities are markers for abnormal cell divisions and cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model

We utilized the transgenic adenocarcinoma mouse prostate (TRAMP) model to study the formation of abnormal mitosis in malignant tumors of the prostate. The results presented here are focused on centrosome and centriole abnormalities and the implications for abnormal cell divisions, genomic instability, and apoptosis. Centrosomes are microtubule organizing organelles which assemble bipolar spindles in normal cells but can organize mono-, tri-, and multipolar mitoses in tumor cells, as shown here with histology and electron microscopy in TRAMP neoplastic tissue. These abnormalities will cause unequal distribution of chromosomes and can initiate imbalanced cell cycles in which checkpoints for cell cycle control are lost. Neoplastic tissue of the TRAMP model is also characterized by numerous apoptotic cells. This may be the result of multipolar mitoses related to aberrant centrosome formations. Our results also reveal that centrosomes at the poles in mitotic cancer cells contain more than the regular perpendicular pair of centrioles which indicates abnormal distribution of centrioles during separation to the mitotic poles. Abnormalities in the centriole-centrosome complex are also seen during interphase where the complex is either closely associated with the nucleus or loosely dispersed in the cytoplasm. An increase in centriole numbers is observed during interphase, which may be the result of increased centriole duplication. Alternatively, these centrioles may be derived from basal bodies that have accumulated in the cell's cytoplasm, after the loss of cell borders. The supernumerary centrioles may participate in the formation of abnormal mitoses during cell division. These results demonstrate multiple abnormalities in the centrosome-centriole complex during prostate cancer that result in abnormal mitoses and may lead to increases in genomic instability and/or apoptosis.     

From Zero to Many: Control of Centriole Number in Development and Disease

Centrioles are essential for the formation of microtubule-derived structures, including cilia, flagella and centrosomes. These structures are involved in a variety of functions, from cell motility to division. In most dividing animal cells, centriole formation is coupled to the chromosome cycle. However, this is not the case in certain specialized divisions, such as meiosis, and in some differentiating cells. For example, oocytes loose their centrioles upon differentiation, whereas multiciliated epithelial cells make several of those structures after they exit the cell cycle. Aberrations of centriole number are seen in many cancer cells. Recent studies began to shed light on the molecular control of centriole number, its variations in development, and how centriole number changes in human disease. Here we review the recent developments in this field.     

Cnn dynamics drive centrosome size asymmetry to ensure daughter centriole retention in Drosophila neuroblasts

Centrosomes comprise a pair of centrioles surrounded by an amorphous network of pericentriolar material (PCM). In certain stem cells, the two centrosomes differ in size, and this appears to be important for asymmetric cell division [1, 2]. In some cases, centrosome asymmetry is linked to centriole age because the older, mother centriole always organizes more PCM than the daughter centriole, thus ensuring that the mother centriole is always retained in the stem cell after cell division [3]. This has raised the possibility that an "immortal" mother centriole may help maintain stem cell fate [4, 5]. It is unclear, however, how centrosome size asymmetry is generated in stem cells. Here we provide compelling evidence that centrosome size asymmetry in Drosophila neuroblasts is generated by the differential regulation of Cnn incorporation into the PCM at mother and daughter centrioles. Shortly after centriole separation, mother and daughter centrioles organize similar amounts of PCM, but Cnn incorporation is then rapidly downregulated at the mother centriole, while it is maintained at the daughter centriole. This ensures that the daughter centriole maintains its PCM and so its position at the apical cortex. Thus, the?daughter centriole, rather than an "immortal" mother centriole, is ultimately retained in these stem cells.     

Centrobin-Centrosomal Protein 4.1-associated Protein (CPAP) Interaction Promotes CPAP Localization to the Centrioles during Centriole Duplication

Centriole duplication is the process by which two new daughter centrioles are generated from the proximal end of preexisting mother centrioles. Accurate centriole duplication is important for many cellular and physiological events, including cell division and ciliogenesis. Centrosomal protein 4.1-associated protein (CPAP), centrosomal protein of 152 kDa (CEP152), and centrobin are known to be essential for centriole duplication. However, the precise mechanism by which they contribute to centriole duplication is not known. In this study, we show that centrobin interacts with CEP152 and CPAP, and the centrobin-CPAP interaction is critical for centriole duplication. Although depletion of centrobin from cells did not have an effect on the centriolar levels of CEP152, it caused the disappearance of CPAP from both the preexisting and newly formed centrioles. Moreover, exogenous expression of the CPAP-binding fragment of centrobin also caused the disappearance of CPAP from both the preexisting and newly synthesized centrioles, possibly in a dominant negative manner, thereby inhibiting centriole duplication and the PLK4 overexpression-mediated centrosome amplification. Interestingly, exogenous overexpression of CPAP in the centrobin-depleted cells did not restore CPAP localization to the centrioles. However, restoration of centrobin expression in the centrobin-depleted cells led to the reappearance of centriolar CPAP. Hence, we conclude that centrobin-CPAP interaction is critical for the recruitment of CPAP to procentrioles to promote the elongation of daughter centrioles and for the persistence of CPAP on preexisting mother centrioles. Our study indicates that regulation of CPAP levels on the centrioles by centrobin is critical for preserving the normal size, shape, and number of centrioles in the cell.     

Centrin-2 is required for centriole duplication in mammalian cells

BACKGROUND: Centrosomes are the favored microtubule-organizing framework of eukaryotic cells. Centrosomes contain a pair of centrioles that normally duplicate once during the cell cycle to give rise to two mitotic spindle poles, each containing one old and one new centriole. However, aside from their role as an anchor point for pericentriolar material and as basal bodies of flagella and cilia, the functional attributes of centrioles remain enigmatic. RESULTS: Here, using RNA interference, we demonstrate that "knockdown" of centrin-2, a protein of centrioles, results in failure of centriole duplication during the cell cycle in HeLa cells. Following inhibition of centrin-2 synthesis, the preexisting pair of centrioles separate, and functional bipolar spindles form with only one centriole at each spindle pole. Centriole dilution results from the ensuing cell division, and daughter cells are "born" with only a single centriole. Remarkably, these unicentriolar daughter cells may complete a second and even third bipolar mitosis in which spindle microtubules converge onto unusually broad spindle poles and in which cell division results in daughter cells containing either one or no centrioles at all. Cells thus denuded of the mature or both centrioles fail to undergo cytokinesis in subsequent cell cycles, give rise to multinucleate products, and finally die. CONCLUSIONS: These results demonstrate a requirement for centrin in centriole duplication and demonstrate that centrioles play a role in organizing spindle pole morphology and in the completion of cytokinesis.     

INDEPENDENCE OF CENTRIOLE FORMATION AND DNA SYNTHESIS

The temporal relationship between cell cycle events and centriole duplication was investigated electron microscopically in L cells synchronized by mechanically selecting mitotic cells. The two mature centrioles which each cell received at telophase migrated together from the side of the telophase nucleus distal to the stem body around to a region of the cytoplasm near the stem body and then into a groovelike indention in the early G₁ nucleus, where they were found throughout interphase. Procentrioles appeared in association with each mature centriole at times varying from 4 to 12 h after mitosis. Since S phase was found to begin on the average about 9 h after mitotic selection, it appeared that cells generated procentrioles late in G₁ or early in S. During prophase, the two centriolar duplexes migrated to opposite sides of the nucleus and the daughter centrioles elongated to the mature length. To ascertain whether any aspect of centriolar duplication was contingent upon nuclear DNA synthesis, arabinosyl cytosine was added to mitotic cells at a concentration which inhibited cellular DNA synthesis by more than 99%. Though cells were thus prevented from entering S phase, the course of procentriole formation was not detectibly affected. However, cells were inhibited from proceeding to the next mitosis, and the centriolar elongation and migration normally associated with prophase did not occur.     

The mother centriole plays an instructive role in defining cell geometry

Centriole positioning is a key step in establishment and propagation of cell geometry, but the mechanism of this positioning is unknown. The ability of pre-existing centrioles to induce formation of new centrioles at a defined angle relative to themselves suggests they may have the capacity to transmit spatial information to their daughters. Using three-dimensional computer-aided analysis of cell morphology in Chlamydomonas, we identify six genes required for centriole positioning relative to overall cell polarity, four of which have known sequences. We show that the distal portion of the centriole is critical for positioning, and that the centriole positions the nucleus rather than vice versa. We obtain evidence that the daughter centriole is unable to respond to normal positioning cues and relies on the mother for positional information. Our results represent a clear example of "cytotaxis" as defined by Sonneborn, and suggest that centrioles can play a key function in propagation of cellular geometry from one generation to the next. The genes documented here that are required for proper centriole positioning may represent a new class of ciliary disease genes, defects in which would be expected to cause disorganized ciliary position and impaired function.