Cytoskeletal organization of a cloned hemopoietic stromal cell line during attachment and spreading

The cell membrane of a cloned murine bone marrow stromal cell line D2XRII was extracted in situ using Triton X-100 detergent and the cytoskeletal structure studied during the process of adherence and spreading. During this process, three zones can be identified in the cytoplasm: the perinuclear zone, which was the fixed part of the cell; the peripheral mixed filamentous zone, which formed the core of long cytoplasmic projections; and an outer zone, which formed the boundary of cytoplasmic projections and contained only intermediate filaments. The process of spreading appeared to originate from very long strips of microfilaments emanating from the second zone, crossing the width of the outer zone, and extending beyond for a long distance. The second and third zones then appeared to "stream out" around the axis of this strip, and in this fashion the cytoplasm spreads over the substratum.     

Cytoskeletal organization: whirling to the beat

Dense populations of microtubules driven by axonemal dynein form large vortices, providing insights into how simple interactions between individuals can give rise to large-scale coordinated movement, such as that seen in schools of fish and flocks of birds.     

Halting a cellular production line: responses to ribosomal pausing during translation

Cellular protein synthesis is a complex polymerization process carried out by multiple ribosomes translating individual mRNAs. The process must be responsive to rapidly changing conditions in the cell that could cause ribosomal pausing and queuing. In some circumstances, pausing of a bacterial ribosome can trigger translational abandonment via the process of trans-translation, mediated by tmRNA (transfer-messenger RNA) and endonucleases. Together, these factors release the ribosome from the mRNA and target the incomplete polypeptide for destruction. In eukaryotes, ribosomal pausing can initiate an analogous process carried out by the Dom34p and Hbs1p proteins, which trigger endonucleolytic attack of the mRNA, a process termed mRNA no-go decay. However, ribosomal pausing can also be employed for regulatory purposes, and controlled translational delays are used to help co-translational folding of the nascent polypeptide on the ribosome, as well as a tactic to delay translation of a protein while its encoding mRNA is being localized within the cell. However, other responses to pausing trigger ribosomal frameshift events. Recent discoveries are thus revealing a wide variety of mechanisms used to respond to translational pausing and thus regulate the flow of ribosomal traffic on the mRNA population.     

Halting a cellular production line: responses to ribosomal pausing during translation.

Cellular protein synthesis is a complex polymerization process carried out by multiple ribosomes translating individual mRNAs. The process must be responsive to rapidly changing conditions in the cell that could cause ribosomal pausing and queuing. In some circumstances, pausing of a bacterial ribosome can trigger translational abandonment via the process of trans-translation, mediated by tmRNA (transfer-messenger RNA) and endonucleases. Together, these factors release the ribosome from the mRNA and target the incomplete polypeptide for destruction. In eukaryotes, ribosomal pausing can initiate an analogous process carried out by the Dom34p and Hbs1p proteins, which trigger endonucleolytic attack of the mRNA, a process termed mRNA no-go decay. However, ribosomal pausing can also be employed for regulatory purposes, and controlled translational delays are used to help co-translational folding of the nascent polypeptide on the ribosome, as well as a tactic to delay translation of a protein while its encoding mRNA is being localized within the cell. However, other responses to pausing trigger ribosomal frameshift events. Recent discoveries are thus revealing a wide variety of mechanisms used to respond to translational pausing and thus regulate the flow of ribosomal traffic on the mRNA population.     

Cytoskeletal organization of limulus amebocytes pre- and post-activation: comparative aspects

One of the major functions of circulating Limulus amebocytes is to effect blood coagulation upon receipt of appropriate signals. However, the hypothesis that Limulus amebocytes are fundamentally similar to vertebrate thrombocytes and platelets has not been tested sufficiently in previous studies of their cytoskeletal organization. Whereas the earlier data were derived from transmission electron microscopy (TEM) of thin sections of a limited number of cells, improved fluorescence labeling methods that retain cell morphology have now enabled us to survey F-actin and microtubule organization in intact individual amebocytes and in large amebocyte populations pre- and post-activation. Anti-tubulin immunofluorescence showed the marginal band (MB) of microtubules to be ellipsoidal in most unactivated cells, with essentially no other microtubules present. However, minor subpopulations of cells with discoidal or pointed shape, containing corresponding arrangements of microtubules suggestive of morphogenetic intermediates, were also observed. Texas-red phalloidin labeled an F-actin-rich cortex in unactivated amebocytes, accounting for MB and granule separation from the plasma membrane as visualized in TEM thin sections, and supporting earlier models for MB maintenance of flattened amebocyte morphology by pressure against a cortical layer. Shape transformation after activation by bacterial lipopolysaccharide was attributable principally to spiky and spreading F-actin in outer cell regions, with the MB changing to twisted, nuclei-associated forms and eventually becoming unrecognizable. These major pre- and post-activation cytoskeletal features resemble those of platelets and non-mammalian vertebrate thrombocytes, supporting recognition of the Limulus amebocyte as a representative evolutionary precursor of more specialized clotting cell types.     

Preventing oxidation of cellular XRCC1 affects PARP-mediated DNA damage responses

Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase β (pol β) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol β in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol β is found in vivo, and it is known that pol β and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C12–C20 oxidized form of XRCC1 and the interaction with pol β, we characterized cell lines representing stable transfectants in Xrcc1^?/? mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12–C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol β (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol β in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.     

Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone

Actions of cytochalasin B (CB) on cytoskeletons and motility of growth cones from cultured Aplysia neurons were studied using a rapid flow perfusion chamber and digital video light microscopy. Living growth cones were observed using differential interference contrast optics and were also fixed at various time points to assay actin filament (F-actin) and microtubule distributions. Treatment with CB reversibly blocked motility and eliminated most of the phalloidin-stainable F-actin from the leading lamella. The loss of F-actin was nearly complete within 2-3 min of CB application and was largely reversed within 5-6 min of CB removal. The loss and recovery of F-actin were found to occur with a very distinctive spatial organization. Within 20-30 s of CB application, F-actin networks receded from the entire peripheral margin of the lamella forming a band devoid of F-actin. This band widened as F-actin receded at rates of 3-6 microns/min. Upon removal of CB, F-actin began to reappear within 20-30 s. The initial reappearance of F-actin took two forms: a coarse isotropic matrix of F-actin bundles throughout the lamella, and a denser matrix along the peripheral margin. The denser peripheral matrix then expanded in width, extending centrally to replace the coarse matrix at rates again between 3-6 microns/min. These results suggest that actin normally polymerizes at the leading edge and then flows rearward at a rate between 3-6 microns/min. CB treatment was also observed to alter the distribution of microtubules, assayed by antitubulin antibody staining. Normally, microtubules are restricted to the neurite shaft and a central growth cone domain. Within approximately 5 min after CB application, however, microtubules began extending into the lamellar region, often reaching the peripheral margin. Upon removal of CB, the microtubules were restored to their former central localization. The timing of these microtubule redistributions is consistent with their being secondary to effects of CB on lamellar F-actin.     

Spy1 expression prevents normal cellular responses to DNA damage: inhibition of apoptosis and checkpoint activation

Spy1 is the originally identified member of the Speedy/Ringo family of vertebrate cell cycle regulators, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Here we report a role for Spy1 in apoptosis and checkpoint activation in response to UV irradiation. Using an inducible system allowing for regulated expression of Spy1, we show that Spy1 expression prevents activation of caspase-3 and suppresses apoptosis in response to UV irradiation. Spy1 expression also allows for UV irradiation-resistant DNA synthesis and permits cells to progress into mitosis, as demonstrated by phosphorylation on histone H3, indicating that Spy1 expression can inhibit the S-phase/replication and G2/M checkpoints. We demonstrate that Spy1 expression inhibits phosphorylation of Chk1, RPA, and histone H2A.X, which may directly contribute to the decrease in apoptosis and checkpoint bypass. Furthermore, mutation of the conserved Speedy/Ringo box, known to mediate interaction with CDK2, abrogates the ability of Spy1 to inhibit apoptosis and the phosphorylation of Chk1 and RPA. The data presented indicate that Spy1 expression allows cells to evade checkpoints and apoptosis and suggest that Spy1 regulation of CDK2 is important for the response to DNA damage.     

Enterococcus faecalis plasmid pAD1-encoded Fst toxin affects membrane permeability and alters cellular responses to lantibiotics

Fst is a peptide toxin encoded by the par toxin-antitoxin stability determinant of Enterococcus faecalis plasmid pAD1. Intracellular overproduction of Fst resulted in simultaneous inhibition of all cellular macromolecular synthesis concomitant with cell growth inhibition and compromised the integrity of the cell membrane. Cells did not lyse or noticeably leak intracellular contents but had specific defects in chromosome partitioning and cell division. Extracellular addition of synthetic Fst had no effect on cell growth. Spontaneous Fst-resistant mutants had a phenotype consistent with changes in membrane composition. Interestingly, overproduction of Fst sensitized cells to the lantibiotic nisin, and Fst-resistant mutants were cross-resistant to nisin and the pAD1-encoded cytolysin.     

Two sides of a cellular coin: CD4(+)CD3- cells regulate memory responses and lymph-node organization

We propose that CD4(+)CD3(-) cells have two functions: a well-established role in organizing lymphoid tissue during development, and a newly discovered role in supporting T-cell help for B cells both during affinity maturation in germinal centres and for memory antibody responses. As CD4(+)CD3(-) cells express the HIV co-receptors CD4 and CXC-chemokine receptor 4, we think that infection of these cells by HIV, and their subsequent destruction by the host immune system, could help to explain the loss of memory antibody responses and the destruction of lymphoid architecture that occur during disease progression to AIDS.     

Cytoskeletal and nuclear organization in mouse embryos derived from nuclear transfer and ICSI: a comparison of agamogony and syngamy before and during the first cell cycle

In this study, somatic cell nuclear transfer (SCNT) and intracytoplasmic sperm injection (ICSI) are used as models of agamogony and syngamy, respectively. In order to elucidate the reasons of low efficiency of somatic cell cloning, cytoskeletal and nuclear organization in cloned mouse embryos was monitored before and during the first cell cycle, and compared with the pattern of ICSI zygote. A metaphase-like spindle with alignment of condensed donor chromosomes was assembled within 3 hr after NT, followed by formation of pronuclear-like structures at 3-6 hr after activation, indicating that somatic nuclear remodeling depends on microtubular network organization. The percentage of two (pseudo-) pronuclei in cloned embryos derived from delayed activation was greater than that in immediate activation group (68.5% vs. 30.8%, P<0.01), but similar to that of ICSI group (68.5% vs. 65.5%, P>0.05). The 2-cell rate in NT embryos was significantly lower than that in zygotes produced by ICSI (64.8% vs. 82.5%, P<0.01). Further studies testified that the cloned embryos reached the metaphase of the first mitosis 10 hr after activation, whereas this occurred at 18 hr in the ICSI zygotes. Comparision of the pattern of microfilament assembly in early NT embryos with that in syngamic zygotes suggested that abnormal microfilamental pattern in cloned embryos may threaten subsequent embryonic development. In conclusion, agamogony, in contrast to syngamy, displays some unique features in respect of cytoskeletal organization, the most remarkable of which is that the first cell cycle is initiated ahead distinctly, which probably leads to incomplete organization of the first mitotic spindle, and contributes to low efficiency of cloning.     

ATR: a master conductor of cellular responses to DNA replication stress

The integrity of the genome is constantly challenged by intrinsic and extrinsic genotoxic stresses that damage DNA. The cellular responses to DNA damage are orchestrated by DNA damage signaling pathways, also known as DNA damage checkpoints. These signaling pathways play crucial roles in detecting DNA damage, regulating DNA repair and coordinating DNA repair with other cellular processes. In vertebrates, the ATM- and Rad3-related (ATR) kinase plays a key role in the response to a broad spectrum of DNA damage and DNA replication stress. Here, we will discuss the recent findings on how ATR is activated by DNA damage and how it protects the genome against interference with DNA replication.     

Insulin responses to mixed meals: comparison of an artificial beta cell and normal beta cells.

The peripheral glucose and free insulin levels seen following a mixed meal in six insulin-dependent diabetic patients whose insulin was administered by a glucose-controlled insulin infusion system (GCIIS) were compared to those of normal subjects who received the same mixed meal or who were given separately carbohydrate, protein, or fat in amounts equivalent to those contained in the mixed meal. Patients treated with the GCIIS achieved nearly normal glucose levels immediately after the mixed meal, but this was accompanied by marked hyperinsulinemia. In the period from 120 to 240 minutes after the start of the mixed meal, the GCIIS duplicates the insulin levels produced by the normal pancreas after a glucose meal and, with appropriate algorithm constants, closely approximates those seen after a mixed meal.     

Correlation between effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro

To compare the effects of cytochalasins on the cellular level with those on the molecular level, 24 cytochalasins, 20 natural compounds and 4 derivatives, were used. The following effects were tested for each of 24 cytochalasins; (a) four high dose (2-20 muM) effects on the cellular level: rounding up of fibroblastic cells, contraction of actin cables, formation of hairy filaments containing actin, and inhibition of lymphocyte capping; (b) a low dose (0.2-2 muM) effect: inhibition of membrane ruffling; and (c) two in vitro effects: an inhibition of actin filament elongation (the high affinity effect [low dose effect] in vitro) and an effect on viscosity of actin filaments(the low affinity effect [high dose effect] in vitro). These results indicated that there are almost the same hierarchic orders of relative effectiveness of different cytochalasins between low and high dose effects and between cellular and molecular effects. From the data obtained with the 24 cytochalasins, we have calculated correlation coefficients of 0.87 and 0.79 between an effect in vivo, inhibition of capping, and an effect in vitro, inhibition of actin filament elongation, as well as between inhibition of capping and another effect in vitro, effect on viscosity of actin filaments, respectively. Furthermore, a correlation coefficient between the high affinity effect and the low affinity effect determined in vitro was calculated to be 0.90 from the data obtained in this study. The strong positive correlation among low and high dose effects in vivo and those in vitro suggests that most of the effects caused by a cytochalasin, irrespective of doses or affected phenomena, might be attributed to the interaction between the drug and the common target protein, actin. In the course of the immunofluorescence microscope study on cytochalasin-treated cells using actin antibody, we have found that aspochalasin D, a 10-isopropylcytochalasin, strongly induced the formation of rodlets containing actin in the cytoplasm of the treated fibroblasts. In contrast, the other cytochalasins, including cytochalasin B, cytochalasin C, cytochalasin D, and cytochalasin H, were found to induce the formation of nuclear rodlets. Both cytoplasmic and nuclear rodlets found in the cytochalasin-treated cells were similar in ultrastructures to those induced by 5 to 10 percent (vol/vol) dimethyl sulfoxide in the same type of cells.     

ATM and ATR: networking cellular responses to DNA damage

Maintenance of genome stability depends on the appropriate response to DNA damage. This response is based on complex networks of signaling pathways that activate numerous processes and lead ultimately to damage repair and cellular survival - or apoptosis. The protein kinases ATM and ATR are master controllers of some of these networks, acting either in concert or separately to orchestrate the responses to specific types of DNA damage or stalled replication. Understanding their mode of action is essential to our understanding of how cells cope with genotoxic stress.     

Generic signal-specific responses: cytokinin and context-dependent cellular responses

The phytohormone cytokinin triggers numerous and diverse responses during the plant life cycle via a two-component phosphorelay signalling system. Each step of the signalling cascade is supported by a gene family comprising several members. While functional redundancy is observed among family members, additional gene-specific functions encoded by cis-regulatory and coding sequence of individual family members have been described and contribute to specificity in signalling output. In addition, the cellular context of the signal-receiving cell affects the response triggered. Recent studies in Arabidopsis have demonstrated how expression of cytokinin signalling components predefines a spatiotemporal map of signalling sensitivity, which causes local signal amplification and attenuation. In summary, the specific interpretation of cytokinin signalling is affected by an orchestrated interplay of signalling genes and cellular context.     

Experiments inside a box lead to out-of-the-box ideas on cellular organization

Microtubules are biopolymers that assemble from tubulin dimers into hollow tubes and play an important role in cellular organization. Their fascinating properties and variety of functions, like for example chromosome segregation, sperm propagation and polarity establishment, have made them a popular subject of study. In this perspective I focus on the contribution of minimal in vitro systems to our understanding of microtubule organization within the physical confinement of a cell.