No elevated cell surface charge at mitosis in X-ray-irradiated mammalian cells

Rounded mitotic cells showed 30% enhanced electrophoretic mobility (EPM) when compared to spindle-formed interphase cells. This increase in EPM that was not present in interphase cells that had been rounded chemically by EDTA is considered to reflect a structural change in the cell membrane during mitosis. X-ray irradiation induced a dose-dependent EPM decrease in both interphase and mitotic cells during a 4-hour period. During the next 20 h of incubation, EPM recovery took place in cells irradiated with 250R, but not in cells exposed to 1000R. EPM was enhanced during mitosis in cells irradiated with low doses, but was absent in cells irradiated with 1000R. The ratio of colony-forming cells and of electrophoretically recovered mitotic cells after 24 h of exposure showed a good statistical correlation. These results indicate that unrepaired membrane damage contributes to mitotic cell death after irradiation.     

DNA damage signaling in response to double-strand breaks during mitosis

The signaling cascade initiated in response to DNA double-strand breaks (DSBs) has been extensively investigated in interphase cells. Here, we show that mitotic cells treated with DSB-inducing agents activate a “primary” DNA damage response (DDR) comprised of early signaling events, including activation of the protein kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), histone H2AX phosphorylation together with recruitment of mediator of DNA damage checkpoint 1 (MDC1), and the Mre11–Rad50–Nbs1 (MRN) complex to damage sites. However, mitotic cells display no detectable recruitment of the E3 ubiquitin ligases RNF8 and RNF168, or accumulation of 53BP1 and BRCA1, at DSB sites. Accordingly, we found that DNA-damage signaling is attenuated in mitotic cells, with full DDR activation only ensuing when a DSB-containing mitotic cell enters G1. Finally, we present data suggesting that induction of a primary DDR in mitosis is important because transient inactivation of ATM and DNA-PK renders mitotic cells hypersensitive to DSB-inducing agents.     

Chromosome Architecture and Genome Organization

How the same DNA sequences can function in the three-dimensional architecture of interphase nucleus, fold in the very compact structure of metaphase chromosomes and go precisely back to the original interphase architecture in the following cell cycle remains an unresolved question to this day. The strategy used to address this issue was to analyze the correlations between chromosome architecture and the compositional patterns of DNA sequences spanning a size range from a few hundreds to a few thousands Kilobases. This is a critical range that encompasses isochores, interphase chromatin domains and boundaries, and chromosomal bands. The solution rests on the following key points: 1) the transition from the looped domains and sub-domains of interphase chromatin to the 30-nm fiber loops of early prophase chromosomes goes through the unfolding into an extended chromatin structure (probably a 10-nm “beads-on-a-string” structure); 2) the architectural proteins of interphase chromatin, such as CTCF and cohesin sub-units, are retained in mitosis and are part of the discontinuous protein scaffold of mitotic chromosomes; 3) the conservation of the link between architectural proteins and their binding sites on DNA through the cell cycle explains the “mitotic memory” of interphase architecture and the reversibility of the interphase to mitosis process. The results presented here also lead to a general conclusion which concerns the existence of correlations between the isochore organization of the genome and the architecture of chromosomes from interphase to metaphase.     

Cell cycle age dependence for radiation-induced G2 arrest: evidence for time-dependent repair

Exponentially growing eucaryotic cells, irradiated in interphase, are delayed in progression to mitosis chiefly by arrest in G2. The sensitivity of Chinese hamster ovary cells to G2-arrest induction by X rays increases through the cell cycle, up to the X-ray transition point (TP) in G2. This age response can be explained by cell cycle age-dependent changes in susceptibility of the target(s) for G2 arrest and/or by changes in capability for postirradiation recovery from G2-arrest damage. Discrimination between sensitivity changes and repair phenomena is possible only if the level of G2-arrest-causing damage sustained by a cell at the time of irradiation and the level ultimately expressed as arrest can be determined. The ability of caffeine to ameliorate radiation-induced G2 arrest, while inhibiting repair of G2-arrest-causing damage makes such an analysis possible. CHO cell monolayers were irradiated (1.5 Gy), then exposed to 5 mM caffeine for periods of 0-10 hr. Cell progression was monitored by the mitotic cell selection procedure. In the presence of caffeine, progression of irradiated cells was relatively unperturbed, but on caffeine removal, G2 arrest was expressed. The duration of G2 arrest was independent of the length of the prior caffeine exposure and, since cells of all ages were ultimately examined, the duration of arrest was also independent of cell cycle age at the time of irradiation. This finding indicates that the target for G2-arrest induction is present throughout the cell cycle and that the level of G2-arrest damage incurred is initially constant for all cell cycle phases. The data are consistent with the existence of a time-dependent recovery mechanism to explain the age dependence for radiation induction of G2 arrest.     

Computerized video time lapse study of cell cycle delay and arrest, mitotic catastrophe, apoptosis and clonogenic survival in irradiated 14-3-3sigma and CDKN1A (p21) knockout cell lines

Computerized video time lapse (CVTL) microscopy was used to observe cellular events induced by ionizing radiation (10-12 Gy) in nonclonogenic cells of the wild-type HCT116 colorectal carcinoma cell line and its three isogenic derivative lines in which p21 (CDKN1A), 14-3-3sigma or both checkpoint genes (double-knockout) had been knocked out. Cells that fused after mitosis or failed to complete mitosis were classified together as cells that underwent mitotic catastrophe. Seventeen percent of the wild-type cells and 34-47% of the knockout cells underwent mitotic catastrophe to enter generation 1 with a 4N content of DNA, i.e., the same DNA content as irradiated cells arrested in G(2) at the end of generation 0. Radiation caused a transient division delay in generation 0 before the cells divided or underwent mitotic catastrophe. Compared with the division delay for wild-type cells that express CDKN1A and 14-3-3sigma, knocking out CDKN1A reduced the delay the most for cells irradiated in G(1) (from approximately 15 h to approximately 3- 5 h), while knocking out 14-3-3sigma reduced the delay the most for cells irradiated in late S and G(2) (from approximately 18 h to approximately 3-4 h). However, 27% of wild-type cells and 17% of 14-3-3sigma(-/-) cells were arrested at 96 h in generation 0 compared with less than 1% for CDKN1A(-/-) and double-knockout cells. Thus expression of CDKN1A is necessary for the prolonged delay or arrest in generation 0. Furthermore, CDKN1A plays a crucial role in generation 1, greatly inhibiting progression into subsequent generations of both diploid cells and polyploid cells produced by mitotic catastrophe. Thus, in CDKN1A-deficient cell lines, a series of mitotic catastrophe events occurred to produce highly polyploid progeny during generations 3 and 4. Most importantly, the polyploid progeny produced by mitotic catastrophe events did not die sooner than the progeny of dividing cells. Death was identified as loss of cell movement, i.e. metabolic activity. Thus mitotic catastrophe itself is not a direct mode of death. Instead, apoptosis during interphase of both uninucleated and polyploid cells was the primary mode of death observed in the four cell types. Knocking out either CDKN1A or 14-3-3sigma increased the amount of cell death at 96 h, from 52% to approximately 70%, with an even greater increase to 90% when both genes were knocked out. Thus, in addition to effects of CDKN1A and 14-3-3sigma expression on transient cell cycle delay, CDKN1A has both an anti-proliferative and anti-apoptosis function, while 14-3-3sigma has only an anti-apoptosis function. Finally, the large alterations in the amounts of cell death did not correlate overall with the small alterations in clonogenic survival (dose-modifying ratios of 1.05-1.13); however, knocking out CDKN1A resulted in a decrease in arrested cells and an increase in survival, while knocking out 14-3-3sigma resulted in an increase in apoptosis and a decrease in survival.     

Entry into Mitosis in Vertebrate Somatic Cells Is Guarded by a Chromosome Damage Checkpoint That Reverses the Cell Cycle When Triggered during Early but Not Late Prophase

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G₂ checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.     

Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-alpha tubulin

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-alpha tubulin construct and a cell line permanently expressing GFP-alpha tubulin was established (LLCPK-1alpha). The mitotic index and doubling time for LLCPK-1alpha were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1alpha cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1alpha and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1alpha cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.     

DNA Damage to a Single Chromosome End Delays Anaphase Onset

Chromosome ends contain nucleoprotein structures known as telomeres. Damage to chromosome ends during interphase elicits a DNA damage response (DDR) resulting in cell cycle arrest. However, little is known regarding the signaling from damaged chromosome ends (designated here as “TIPs”) during mitosis. In the present study, we investigated the consequences of DNA damage induced at a single TIP in mitosis. We used laser microirradiation to damage mitotic TIPs or chromosome arms (non-TIPs) in PtK2 kidney epithelial cells. We found that damage to a single TIP, but not a non-TIP, delays anaphase onset. This TIP-specific checkpoint response is accompanied by differential recruitment of DDR proteins. Although phosphorylation of H2AX and the recruitment of several repair factors, such as Ku70-Ku80, occur in a comparable manner at both TIP and non-TIP damage sites, DDR factors such as ataxia telangiectasia mutated (ATM), MDC1, WRN, and FANCD2 are specifically recruited to TIPs but not to non-TIPs. In addition, Nbs1, BRCA1, and ubiquitin accumulate at damaged TIPs more rapidly than at damaged non-TIPs. ATR and 53BP1 are not detected at either TIPs or non-TIPs in mitosis. The observed delay in anaphase onset is dependent on the activity of DDR kinases ATM and Chk1, and the spindle assembly checkpoint kinase Mps1. Cells damaged at a single TIP or non-TIP eventually exit mitosis with unrepaired lesions. Damaged TIPs are segregated into micronuclei at a significantly higher frequency than damaged non-TIPs. Together, these findings reveal a mitosis-specific DDR uniquely associated with chromosome ends.     

Haspin inhibition delays cell cycle progression through interphase in cancer cells

Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) is a serine/threonine kinase pertinent to normal mitosis progression and mitotic phosphorylation of histone H3 at threonine 3 in mammalian cells. Different classes of small molecule inhibitors of haspin have been developed and utilized to investigate its mitotic functions. We report herein that applying haspin inhibitor CHR-6494 or 5-ITu at the G1/S boundary could delay mitotic entry in synchronized HeLa and U2OS cells, respectively, following an extended G2 or the S phase. Moreover, late application of haspin inhibitors at S/G2 boundary is sufficient to delay mitotic onset in both cell lines, thereby, indicating a direct effect of haspin on G2/M transition. A prolonged interphase duration is also observed with knockdown of haspin expression in synchronized and asynchronous cells. These results suggest that haspin can regulate cell cycle progression at multiple stages at both interphase and mitosis.     

Time-Lapse Cinemicrographic Studies of X-Irradiated HeLa S3 Cells: II. Cell Fusion

Analysis of time-lapse cinemicrographs of X-irradiated HeLa S3 cells has shown that the incidence of cell fusion was increased from 0.9% (following 1267 divisions) in control cells to an average of 22% (following 655 divisions) in cells irradiated with 500 rad doses of 220 kv X-rays. The incidence depended on the stage of the generation cycle at which the parent cells were irradiated. It was nearly constant in the first three postirradiation generations. Fusion occurred at all stages of the generation cycle, but preferentially during the first 20%. Cells undergoing fusion progressed more slowly through the generation cycle and had a higher probability of disintegrating than did irradiated cells that did not fuse. The occurrence of fusion was clonally distributed in the population. It took place only between sister (or closely related) cells. Protoplasmic bridges were often visible between sister cells prior to fusion. Giant cells arose only as a result of fusion. The incidence of multipolar divisions, though higher than in unirradiated cells, was only 5.5% in cultures irradiated with 500 rads. Fusion occurred following 85% of the multipolar divisions and was often followed by a multipolar division.     

Cell Cycle–coupled Relocation of Types I and II Topoisomerases and Modulation of Catalytic Enzyme Activities

We visualized DNA topoisomerases in A431 cells and isolated chromosomes by isoenzyme-selective immunofluorescence microscopy. In interphase, topoisomerase I mainly had a homogeneous nuclear distribution. 10–15% of the cells exhibited granular patterns, 30% showed bright intranucleolar patches. Topoisomerase II isoenzymes showed spotted (α) or reticular (β) nuclear patterns throughout interphase. In contrast to topoisomerase IIα, topoisomerase IIβ was completely excluded from nucleoli. In mitosis, topoisomerase IIβ diffused completely into the cytosol, whereas topoisomerases I and IIα remained chromosome bound. Chromosomal staining of topoisomerase I was homogeneous, whereas topoisomerase IIα accumulated in the long axes of the chromosome arms and in the centriols. Topoisomerase antigens were 2–3-fold higher in mitosis than in interphase, but specific activities of topoisomerase I and II were reduced 5- and 2.4-fold, respectively. These changes were associated with mitotic enzyme hyperphosphorylation. In interphase, topoisomerases could be completely linked to DNA by etoposide or camptothecin, whereas in mitosis, 50% of topoisomerase IIα escaped poisoning. Refractoriness to etoposide could be assigned to the salt-stable scaffold fraction of topoisomerase IIα, which increased from <2% in G₁ phase to 48% in mitosis. Topoisomerases I and IIβ remained completely extractable throughout the cell cycle. In summary, expression of topoisomerases increases towards mitosis, but specific activities decrease. Topoisomerase IIβ is released from the heterochromatin, whereas topoisomerase I and IIα remain chromosome bound. Scaffold-associated topoisomerase IIα appears not to be involved in catalytic DNA turnover, though it may play a role in the replicational cycle of centriols, where it accumulates during M phase.     

Different cyclic changes in the surface membrane of normal and malignant transformed cells

Transformed fibroblasts in interphase and normal fibroblasts in mitosis were agglutinated by Con A and the lectin from wheat germ, whereas normal fibroblasts in interphase and transformed fibroblasts in mitosis were not agglutinated by these lectins. The percentage of fluorescent cells at non-saturation concentrations of fluorescent ConA was also higher with transformed interphase and normal mitotic cells, than with normal interphase and transformed mitotic cells. Under the same conditions, a similar number of radioactively labeled ConA molecules were bound to normal and transformed cells in interphase and mitosis. Our results indicate different cyclic changes in the surface membrane of normal and transformed fibroblasts, so that regarding interaction with these lectins, normal mitotic cells resemble transformed interphase cells and transformed mitotic resemble normal interphase cells. The data suggest that there is a reversed cyclic change in the mobility of specific surface membrane sites in normal and transformed cells.     

REPOPULATION OF THE POSTMITOTIC NUCLEOLUS BY PREFORMED RNA

This study is concerned with the fate of the nucleolar contents, particularly nucleolar RNA, during mitosis Mitotic cells harvested from monolayer cultures of Chinese hamster embryonal cells, KB6 (human) cells, or L929 (mouse) cells were allowed to proceed into interphase in the presence or absence (control) of 0.04–0 08 µg/ml of actinomycin D, a concentration which preferentially inhibits nucleolar (ribosomal) RNA synthesis 3 hr after mitosis, control cells had large, irregularly shaped nucleoli which stained intensely for RNA with azure B and for protein with fast green. In cells which had returned to interphase in the presence of actinomycin D, nucleoli were segregated into two components easily resolvable in the light microscope, and one of these components stained intensely for RNA with azure B. Both nucleolar components stained for protein with fast green In parallel experiments, cultures were incubated with 0.04–0 08 µg/ml actinomycin D for 3 hr before harvesting of mitotic cells, then mitotic cells were washed and allowed to return to interphase in the absence of actinomycin D. 3 hr after mitosis, nuclei of such cells were devoid of large RNA-containing structures, though small, refractile nucleolus-like bodies were observed by phase-contrast microscopy or in material stained for total protein. These experiments indicate that nucleolar RNA made several hours before mitosis persists in the mitotic cell and repopulates nucleoli when they reform after mitosis     

Variants of asynchronous DNA synthesis and mitoses in multinucleate cells induced by cytochalasin B

Cases of asynchronous progression with separate nuclei of S-period and initial mitotic stages in multinucleate cells were discovered in Chinese hamster cell cultures during a prolonged action of cytochalasin B (7 days) and after its stopping (7 days of cell cultivation without drug). The interphase asynchrony under experimental conditions vary in value corresponding to the level of interphase asynchrony in spontaneous multinucleate cells in control cultures. So, the interphase asynchrony in cytochalasin B-induced multinucleate cells is suggested not to be connected with the drug action. Fusion of heterophase cells and a high level of proliferation activity of multinucleate cells seem to be the main reason of interphase asynchrony both in control cultures and in experimental conditions. Unlike the interphase asynchrony, the appearance of the mitotic asynchrony in multinucleate cells is shown to be connected with the action of cytochalasin B. The high level of the mitotic asynchrony remains after the stopping of drug action. A conclusion is made that mitotic asynchrony of nuclei, along with multipolar mitosis and cytokinesis inhibition, is one more display of the cytotoxic action of cytochalasin B on mitosis.     

CDK-dependent phosphorylation of Alp7–Alp14 (TACC–TOG) promotes its nuclear accumulation and spindle microtubule assembly

As cells transition from interphase to mitosis, the microtubule cytoskeleton is reorganized to form the mitotic spindle. In the closed mitosis of fission yeast, a microtubule-associated protein complex, Alp7–Alp14 (transforming acidic coiled-coil–tumor overexpressed gene), enters the nucleus upon mitotic entry and promotes spindle formation. However, how the complex is controlled to accumulate in the nucleus only during mitosis remains elusive. Here we demonstrate that Alp7–Alp14 is excluded from the nucleus during interphase using the nuclear export signal in Alp14 but is accumulated in the nucleus during mitosis through phosphorylation of Alp7 by the cyclin-dependent kinase (CDK). Five phosphorylation sites reside around the nuclear localization signal of Alp7, and the phosphodeficient alp7-5A mutant fails to accumulate in the nucleus during mitosis and exhibits partial spindle defects. Thus our results reveal one way that CDK regulates spindle assembly at mitotic entry: CDK phosphorylates the Alp7–Alp14 complex to localize it to the nucleus.     

Mitotic Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1232 by Cdk1:Cyclin B Inhibits eIF4A Helicase Complex Binding with RNA

During mitosis, global translation is suppressed, while synthesis of proteins with vital mitotic roles must go on. Prior evidence suggests that the mitotic translation shift involves control of initiation. Yet, no signals specifically targeting translation initiation factors during mitosis have been identified. We used phosphoproteomics to investigate the central translation initiation scaffold and “ribosome adaptor,” eukaryotic initiation factor 4G1 (eIF4G1) in interphase or nocodazole-arrested mitotic cells. This approach and kinase inhibition assays, in vitro phosphorylation with recombinant kinase, and kinase depletion-reconstitution experiments revealed that Ser1232 in eIF4G1 is phosphorylated by cyclin-dependent kinase 1 (Cdk1):cyclin B during mitosis. Ser1232 is located in an unstructured region of the C-terminal portion of eIF4G1 that coordinates assembly of the eIF4G/-4A/-4B helicase complex and binding of the mitogen-activated protein kinase (MAPK) signal-integrating kinase, Mnk. Intense phosphorylation of Ser1232 in mitosis strongly enhanced the interactions of eIF4A with HEAT domain 2 of eIF4G and decreased association of eIF4G/-4A with RNA. Our findings implicate phosphorylation of eIF4G1(Ser1232) by Cdk1:cyclin B and its inhibitory effects on eIF4A helicase activity in the mitotic translation initiation shift.     

A Highlights from MBoC Selection: Mitotic regulation of fungal cell-to-cell connectivity through septal pores involves the NIMA kinase

Intercellular bridges are a conserved feature of multicellular organisms. In multicellular fungi, cells are connected directly via intercellular bridges called septal pores. Using Aspergillus nidulans, we demonstrate for the first time that septal pores are regulated to be opened during interphase but closed during mitosis. Septal pore–associated proteins display dynamic cell cycle–regulated locations at mature septa. Of importance, the mitotic NIMA kinase locates to forming septa and surprisingly then remains at septa throughout interphase. However, during mitosis, when NIMA transiently locates to nuclei to promote mitosis, its levels at septa drop. A model is proposed in which NIMA helps keep septal pores open during interphase and then closed when it is removed from them during mitosis. In support of this hypothesis, NIMA inactivation is shown to promote interphase septal pore closing. Because NIMA triggers nuclear pore complex opening during mitosis, our findings suggest that common cell cycle regulatory mechanisms might control septal pores and nuclear pores such that they are opened and closed out of phase to each other during cell cycle progression. The study provides insights into how and why cytoplasmically connected Aspergillus cells maintain mitotic autonomy.     

Computerized video time-lapse (CVTL) analysis of cell death kinetics in human bladder carcinoma cells (EJ30) X-irradiated in different phases of the cell cycle

The purpose of this study was to quantify the modes and kinetics of cell death for EJ30 human bladder carcinoma cells irradiated in different phases of the cell cycle. Asynchronous human bladder carcinoma cells were observed in multiple fields by computerized video time-lapse (CVTL) microscopy for one to two cell divisions before irradiation (6 Gy) and for 6-11 days afterward. By analyzing time-lapse movies collected from these fields, pedigrees were constructed showing the behaviors of 231 cells irradiated in different phases of the cell cycle (i.e. at different times after mitosis). A total of 219 irradiated cells were determined to be non-colony-forming over the time spans of the experiments. In these nonclonogenic pedigrees, cells died primarily by necrosis either without entering mitosis or over 1 to 10 postirradiation generations. A total of 105 giant cells developed from the irradiated cells or their progeny, and 30% (31/105) divided successfully. Most nonclonogenic cells irradiated in mid-S phase (9-12 h after mitosis) died by the second generation, while those irradiated either before or after this short period in mid-S phase had cell deaths occurring over one to nine postirradiation generations. The nonclonogenic cells irradiated in mid-S phase also experienced the longest average delay before their first division. Clonogenic cells (11/12 cells) divided sooner after irradiation than the average nonclonogenic cells derived from the same phase of the cell cycle. The early death and long division delay observed for nonclonogenic cells irradiated in mid-S phase could possibly result from an increase in damage induced during the transition from the replication of euchromatin to the replication of heterochromatin.     

The occurrence and possible role of 80–100 Å filaments in PtKl cells

PtKl cells were examined by surface scanning and transmission electron microscopy to determine the mechanism responsible for the ability of these cells to remain flat during mitosis. Bundles of tightly packed 80–100 Å filaments were seen to radiate from synthesis-and-organizing centers, and to terminate in desmosomes at the plasma membrane. In the presence of Colcemid or colchicine, cells beginning mitosis rounded up and the synthesis-and-organizing centers could no longer be found. In contrast, the occurrence of these structures in interphase cells treated with Colcemid or colchicine was increased. A cytoskeletal role for the 80–100 Å filaments is proposed.