Judith A. Snyder

CONFOCAL ANALYSIS OF PRIMARY CILIA STRUCTURE AND COLOCALIZATION WITH THE GOLGI APPARATUS IN CHONDROCYTES AND AORTIC SMOOTH MUSCLE CELLS

Detyrosinated and acetylated α‐tubulins represent a stable pool of tubulin typically associated with microtubules of the centrosome and primary cilium of eukaryotic cells. Although primary cilium—centrosome and centrosome—Golgi relationships have been identified independently, the precise structural relationship between the primary cilium and Golgi has yet to be specifically defined. Confocal immunohistochemistry was used to localize detyrosinated (ID5) and acetylated (6‐11B‐1) tubulin antibodies in primary cilia of chondrocytes and smooth muscle cells, and to demonstrate their relationship to the Golgi complex identified by complementary lectin staining with wheat germ agglutinin. The results demonstrate the distribution and inherent structural variation of primary cilia tubulins, and the anatomical interrelationship between the primary cilium, the Golgi apparatus and the nucleus. We suggest that these interrelationships may form part of a functional feedback mechanism which could facilitate the directed secretion of newly synthesized connective tissue macromolecules.

CYTOCHALASIN J TREATMENT SIGNIFICANTLY ALTERS MITOTIC SPINDLE MICROTUBULE ORGANIZATION AND KINETOCHORE STRUCTURE IN PTK1 CELLS

It has previously been demonstrated that treatment of mitotic PtK1 cells with 10-20 microg/ml cytochalasin J (CJ) blocks or slows chromosome motion and has a significant effect on spindle architecture [Snyder and Cohen, 1995: Cell Motil. Cytoskeleton 32:245-257]. Spindle microtubules (MTs) were shown to reorganize within the spindle domain, with kinetochore MTs (kMTs) reduced in number and non-kinetochore MTs (nkMTs) shown to splay outside the original spindle domain. In some cases, bundles of MTs were shown to be refocused away from the original spindle poles, creating the appearance of a multi-polar spindle. In this paper we use serial section electron microscopy, coupled with computer-assisted reconstruction techniques, to determine the rearrangement of spindle MTs and chromosome position following brief treatments of mitotic cells with 10-20 microg/ml CJ at various stages of mitosis. CJ treatment of prometaphase cells reduces the number of kMTs and the size and organization of the kinetochore lamina. Instead of kinetochore bundles of MTs aligned parallel to one another and running from kinetochore to pole, this class of MTs is highly fragmented. Non-kinetochore MTs are also highly fragmented, usually less than 2 microm long, and remain relatively straight over short distances, with some MTs arranged at an oblique angle to the longitudinal spindle axis. In approximately 30% of cells treated with CJ, the failure of a small number of chromosomes to attach to spindle fibers can be documented. These chromosomes show a significant change in the organization of the kinetochore laminae. Light microscopic analysis of cells treated with CJ reveals loss of chromosome congression, with chromsomes usually located at the periphery of the spindle and some completely detached from the spindle. Cells treated with 10 microg/ml CJ for 10 min and released into tissue culture medium show a resumption of chromosome motion within a few minutes, both during congression and anaphase. Where kMTs are inserted into kinetochores, chromosome motion is seen; where chromosomes fail to attach to the spindle, no chromosome motion is observed. Cells treated in metaphase show a delayed entry into anaphase and a reduced rate of anaphase A, with the arms of some chromosomes remaining in the interzone region. Our results suggest that CJ-sensitive molecules play a role in the organization of spindle MTs, as well as their functional association to kinetochores.

Analysis of spindle microtubule organization in untreated and taxol-treated PtK1 cells.

Taxol, a microtubule stabilizing agent, has been used to study changes in spindle microtubule organization during mitosis. PtK1 cells have been treated with 5 μg/ml taxol for brief periods to determine its effect on spindle architecture. During prophase taxol induces microtubules to aggregate, particularly evident in the region between the nucleus and cell periphery. Taxol induces astral microtubule formation in prometaphase and metaphase cells concomitant with a reduction in spindle length. At anaphase taxol induces an increase in length in astral microtubules and reduces microtubule length in the interzone. Taxol‐treated telophase cells show a reduction in the rate of furrowing and astral microtubules lack a discrete focus and are arranged more diffusely on the surface of the nuclear envelope. In summary, taxol treatment of cells prior to anaphase produces an increase in astral microtubules, a reduction in kinetochore microtubules and a decrease in spindle length. Brief taxol treatments during anaphase through early G1 promotes stabilization of microtubules, an increase in the length of astral microtubules and a delayed rate of cytokinesis.

Evidence for a ribonucleoprotein complex as a template for microtubule initiation in vivo

At the onset of mitosis in cultured mammalian cells, the centriolar region rapidly initiates the assembly of microtubules (MT) to form two asters which ultimately forms the basis of the mitotic spindle. The rapid change in the centrospheres ability to nucleate MTs at prometaphase maybe due to the presence of an RNase sensitive component. Lysis of colcemid-blocked mitotic cells with RNase A or T2 prior to addition of exogenous microtubule protein greatly diminishes the number of MT that can be nucleated in a lysed cell system. MT initiation can also be reduced or abolished by extended lysis in neutral buffers or brief lysis in acidic buffers. This suggests that the RNA component maybe complexed with a protein to serve as a template for MT initiation at the onset of mitosis.

Anaphase progression and furrow establishment in nocodazole-arrested PtK1 cells.

The relationship between progression through anaphase and furrow establishment was investigated in PtK1 cells using the anti-mitotic agent Nocodazole to arrest cells at different points in anaphase. The capacity of cells to furrow was compared to the kinetochore-kinetochore separation attained at the time of arrest. For the stages of anaphase examined, furrowing capacity increased directly with kinetochore-kinetochore separation until complete furrows were formed after kinetochore-kinetochore separations of 14 μm or more were reached. Furrow establishment thus occurs during a definite interval during anaphase in PtK1 cells. Results from electron microscopy of both Nocodazole-treated and control cells suggest that a population of astral microtubules may be important for furrow establishment.

Localization of myosin II to chromosome arms and spindle fibers in PtK1 cells: a possible role for an actomyosin system in mitosis.

Summary.The enzymes of importance in moving chromosomes are called motor proteins and include dynein, kinesin, and possibly myosin II. These three molecules are all included in the category of ATPases, in that they have the ability to convert chemical energy into mechanical energy. Both dynein and kinesin have been documented as molecules that “walk” along microtubules in the mitotic spindle, carrying cargo such as chromosomes. Myosin II, analogous to the muscle contraction system, transiently interacts along actin filaments and associates with kinetochore microtubules. In this paper we present evidence that a third ATPase, myosin II, may act as a “thruster” to propel chromosomes during the mitotic process. Double-label immunocytochemistry to actin and myosin II shows that myosin II is localized on chromosome arms at the beginning of mitosis and remains localized to the chromosomes throughout mitosis. Specific staining of myosin II is relegated to the outside of chromosomes with the highest density of staining occurring between the spindle poles and the chromosomes. This specific localization could account for the movement of chromosomes during mitosis, since they segregate towards the spindle poles, along kinetochore microtubules containing actin filaments, after aligning at the equatorial region of the cell at metaphase. We conclude from this study that there is an actomyosin system present in the mitotic spindle and that myosin is attached to chromosome arms and may act as a thruster in moving chromosomes during the mitotic process.

Acceleration of cytokinesis in PtK1 cells treated with microtubule inhibitors

Mitotic PtK1 cells were treated at furrow initiation with microtubule poisons to determine the role of microtubules in the regulation of terminal cytokinetic events. The administration of anti-microtubule agents in late anaphase accelerated the rate of cytokinesis by approx. 60% as measured by changes in furrow diameter. Application of colcemid, nocodazole, or vinblastine sulfate to cells at furrow initiation all increased the rate of furrowing. Nocodazole, applied at various concentrations, demonstrated a dose-dependent relationship with furrowing rate. These results suggest a coupling between the disorganization and depolymerization of microtubules and the acceleration of furrowing. Electron microscopic analysis of cells treated with microtubule inhibitors show approx. 70% fewer microtubule profiles in the constricted region between the daughter cells. Microtubules may play a restraining role in the rate of furrowing.

Both actin and myosin inhibitors affect spindle architecture in PtK1 cells: does an actomyosin system contribute to mitotic spindle forces by regulating attachment and movements of chromosomes in mammalian cells?

Immunocytochemical techniques are used to analyze the effects of both an actin and myosin inhibitor on spindle architecture in PtK1 cells to understand why both these inhibitors slow or block chromosome motion and detach chromosomes. Cytochalasin J, an actin inhibitor and a myosin inhibitor, 2, 3 butanedione 2-monoxime, have similar effects on changes in spindle organization. Using primary antibodies and stains, changes are studied in microtubule (MT), actin, myosin, and chromatin localization. Treatment of mitotic cells with both inhibitors results in detachment or misalignment of chromosomes from the spindle and a prominent buckling of MTs within the spindle, particularly evident in kinetochore fibers. Evidence is presented to suggest that an actomyosin system may help to regulate the initial and continued attachment of chromosomes to the mammalian spindle and could also influence spindle checkpoint(s).

An innovative fixative for cytoskeletal components allows high resolution in colocalization studies using immunofluorescence techniques

Using a new fixation solution, CytoSkelFix, it is now possible to obtain superior fixation and thus resolution of cytoskeletal components using immunofluorescence and fluorescence microscopy. This fixative combines rapid cell penetration and cellular crosslinking of proteins such that both preservation and resolution of cellular proteins can be detected. The cytoskeleton has proven very difficult to preserve, partly because of the lability of one of the filament systems (microtubules), and one single fixative is incapable of properly preserving microtubules, microfilaments, and intermediate filaments for localization in the same cell. Further, the motor proteins associated with the cytoskeletal elements are even more difficult to preserve, particularly simultaneously with the fiber system with which they associate. We present evidence that CytoSkelFix is a superior preservative and would be useful in fixation for all types of immunofluorescence colocalization studies where superior preservation is required.

Localization of kinetochore fragments isolated from single chromatids in mitotic CHO cells

SummaryChinese hamster ovary (CHO) cells are treated with hydroxurea followed by a caffeine treatment to form detached kinetochore fragments in the absence of sister chromatids. Detached kinetochores in mitotic CHO cells display a functional association with MTs initiated from one or both centrosomes such that these association(s) have a significant influence on the location and orientation of detached kinetochores and/or their fragments. Kinetochore fragments which are amphitelically oriented are positioned approximately midway between the two centrosomes. Thus, a kinetochore isolated from a single chromatid can capture MTs from both poles. Monotelic orientation of these fragments is more frequently observed with kinetochore fragments located an average distance of 2.5 μm from the nearest centrosome, compared to an average distance of 4.4 μm in amphitelically oriented fragments. In cells treated with the potent MT poison, nocodazole, kinetochore isolation also occurs and therefore is not dependent on the presence of MTs. CHO cells treated to produce isolated kinetochores or kinetochore fragments then subsequently hyperosmotically shocked show no MTs directly inserted into kinetochore lamina, similar to the response of sucrose-treated metapbase PtK1 cells. This treatment shows circular kinetochores tangentially associated with bundles of MTs that are located an average of 1.5 μm from the centrosome. Our results suggest that a single kinetochore fragment can attach to MTs initiated from one or both centrosomes and that their specific association to MT fibers defines orientation of detached kinetochores within the spindle domain.