Functional fluorescently-tagged human profilin

Profilin is an essential regulator of actin and microtubule dynamics and therefore a critical control point for the normal division, motility, and morphology of cells. Most studies of profilin have focused on biochemical investigations using purified protein because high cellular concentrations (121 µM) present challenges for conventional imaging modalities. In addition, past studies that employed direct labeling or conventional fusion protein strategies compromised different facets of profilin function. We engineered a fluorescently-labeled profilin that retains native activities with respect to phosphoinositide lipids, actin monomers, formin-mediated actin assembly, and microtubule polymerization. This fluorescent profilin directly binds to dimers of tubulin (kD = 1.7 µM) and the microtubule lattice (kD = 10 µM) to stimulate microtubule assembly. In cells, our tagged profilin fully rescues profilin-1(-/-) cells from knockout-induced perturbations to cell shape, actin filament architecture, and microtubule arrays. Thus, this labeled profilin-1 is a reliable tool to investigate the dynamic interactions of profilin with actin or microtubules in live cell and in vitro applications.

Coordinating Bacterial Cell Division with Nutrient Availability: a Role for Glycolysis

ABSTRACTCell division in bacteria is driven by a cytoskeletal ring structure, the Z ring, composed of polymers of the tubulin-like protein FtsZ. Z-ring formation must be tightly regulated to ensure faithful cell division, and several mechanisms that influence the positioning and timing of Z-ring assembly have been described. Another important but as yet poorly understood aspect of cell division regulation is the need to coordinate division with cell growth and nutrient availability. In this study, we demonstrated for the first time that cell division is intimately linked to central carbon metabolism in the model Gram-positive bacteriumBacillus subtilis. We showed that a deletion of the gene encoding pyruvate kinase (pyk), which produces pyruvate in the final reaction of glycolysis, rescues the assembly defect of a temperature-sensitiveftsZmutant and has significant effects on Z-ring formation in wild-typeB. subtiliscells. Addition of exogenous pyruvate restores normal division in the absence of the pyruvate kinase enzyme, implicating pyruvate as a key metabolite in the coordination of bacterial growth and division. Our results support a model in which pyruvate levels are coupled to Z-ring assembly via an enzyme that actually metabolizes pyruvate, the E1α subunit of pyruvate dehydrogenase. We have shown that this protein localizes over the nucleoid in a pyruvate-dependent manner and may stimulate more efficient Z-ring formation at the cell center under nutrient-rich conditions, when cells must divide more frequently.IMPORTANCEHow bacteria coordinate cell cycle processes with nutrient availability and growth is a fundamental yet unresolved question in microbiology. Recent breakthroughs have revealed that nutritional information can be transmitted directly from metabolic pathways to the cell cycle machinery and that this can serve as a mechanism for fine-tuning cell cycle processes in response to changes in environmental conditions. Here we identified a novel link between glycolysis and cell division inBacillus subtilis. We showed that pyruvate, the final product of glycolysis, plays an important role in maintaining normal division. Nutrient-dependent changes in pyruvate levels affect the function of the cell division protein FtsZ, most likely by modifying the activity of an enzyme that metabolizes pyruvate, namely, pyruvate dehydrogenase E1α. Ultimately this system may help to coordinate bacterial division with nutritional conditions to ensure the survival of newborn cells.

Growth Inhibition and Disruption of Mitosis by DCPA in Oat (Avena sativa) Roots

One to 5.6 μM DCPA (dimethyl tetrachloroterephthalate) inhibited oat (Avena sativaL. ‘Victory’) root growth within 12 to 18 h. Treated roots were severely stunted and swollen. An analysis of cell division in roots treated with DCPA revealed a disruption of normal mitosis after prophase. Metaphase, anaphase, and telophase division figures were absent 8 to 10 h after treatment with 5.6 μM DCPA. In contrast, a 24-h treatment with 5.6 μM DCPA was necessary to eliminate prophase division figures. The number of aberrant division figures increased concomitantly with the reduction in normal division figures. The predominant type of aberrant division figure was a condensed prophase. When the aberrant division cycle was completed and cells entered interphase, the dispersed chromosomes coalesced to form large, polymorphic nuclei and, occasionally, micronuclei. Approximately 60% of the outer four tiers of cells in roots treated with 5.6 μM DCPA developed abnormal cell walls. These data suggest that DCPA causes root growth inhibition by disrupting several processes involving organized microtubules.

SPONTANEOUS MITOTIC RECOMBINATION AND EVIDENCE FOR AN X-RAY-INDUCIBLE SYSTEM FOR THE REPAIR OF DNA DAMAGE IN DROSOPHILA MELANOGASTER

ABSTRACT Spontaneous mitotic recombination in the left arm of chromosome 3 was examined in both unirradiated control flies and sibs irradiated early in development by determining the sizes and frequencies of multiple-wing-hair (mwh) clones in the wing blade of heterozygous mwh/+ flies. Approximately 16% of the spontaneous mwh clones arise from events generating cells with normal division rates. The remaining 84% result from events generating cells with an average cell division rate one-third that of the surrounding cells; these are thought to result from events that generate aneuploid cells. Such clones probably arise from a failure correctly to repair spontaneous DNA damage. The frequency of spontaneous events late in development decreases significantly after irradiation as much as 150 hours earlier in development. The suppression of spontaneous events decreases with a longer period of time between irradiation and the final cell divisions in the wing blade. These results suggest the existence of a repair system for DNA damage in Drosophila that is induced by irradiation. The decrease in effect with time following irradiation could result from slow degradation or dilution by subsequent cell growth and division.

Cytology of exceptional development of the male gametophyte in Ophiorrhiza mungos

In Ophiorrhiza mungos L. the first division of the microspore nucleus gives rise to a large vegetative nucleus and a smaller generative nucleus. The vegetative nucleus subsequently fragments into a number of irregularly sized particles. These vegetative nuclear bodies move to and flow out of the three germ pores to form three spherical buds on the developing pollen grains. The 'pollen buds' are shed off from the pollen proper before dehiscence. The uninucleate grains readily germinate in vitro, and the single generative nucleus undergoes normal division to give rise to two sperm nuclei.

A FINE STRUCTURAL ANALYSIS OF CLEAVAGE INDUCTION AND FURROWING IN THE EGGS OF ARBACIA PUNCTULATA

A fine structural study has been carried out on the various formed elements present before, during, and after the first cleavage division, not only in normally developing Arbacia eggs, but also in eggs which have been induced to cleave prematurely by high-pressure centrifugation. The aim has been to ascertain whether or not any of the morphologically identifiable components may be involved in initiating the furrowing process. Also, attention has been given to the fine structure of the cytoplasmic cortex, particulary in the walls of the furrow, in the hope of reaching a better understanding of the mechanics of cleavage. The annulate lamellae and the membranous envelope of the nucleus are the only formed elements which disappear shortly before cleavage, not only in eggs undergoing normal division, but also in eggs which have been induced to cleave ahead of schedule by high-pressure, high-force centrifugation. Therefore, it is suggested as a tentative hypothesis that materials liberated upon disintegration of the nuclear membrane and the annulate lamellae play an essential role in initiating and effecting the furrowing reaction, especially since the stratification of these elements in experimentally induced eggs corresponds to the position of the developing furrow. Another of the membranous elements in the egg, the Golgi complex, shows considerable modification as a result of high-pressure centrifugation, but these structures do not undergo disintegration. Rather, they become curled into rounded bodies. The vacuole population is not greatly affected by inducing treatments. During cleavage, both naturally occurring and experimentally induced, a considerable number of 50 A filaments appear in the denser cytoplasmic cortex, but only in the walls of the furrow. These filaments are similar to those which have been demonstrated in a number of contractile cells. Accordingly, it is suggested that this fibrillar system may be actively involved in the development of the cleavage force.

Electrical Signs of New Membrane Production during Cleavage of Rana pipiens Eggs

Rana pipiens eggs dividing normally in diluted Ringer's solution show an increase in transmembrane potential inside negative, a decrease in resistance, and no change in total surface membrane capacitance at the appearance of a division furrow. Furrows of eggs in solutions with the tonicity of full Ringer develop partially, then regress so that the surface is again spherical. The potential and resistance changes are greater and substantial increases in capacitance occur when furrowing is so inhibited. It is proposed that the electrical changes at division are due to the introduction of new plasma membrane, between the blastomeres, having selective permeability to K and a low resistance compared to the outer spherical membrane. A narrow gap between blastomeres limits current flow through new membrane during normal division. A direct exposure of new membrane to the bathing medium when furrowing is disrupted results in larger changes in potential and resistance and permits the capacitance of new membrane to be detected.

MORPHOLOGICAL AND KINETIC ASPECTS OF MITOTIC ARREST BY AND RECOVERY FROM COLCEMID

The effect of Colcemid on the in vivo system of regenerating rat liver and on the in vitro system of HeLa cell cultures was studied to determine some of the morphological and kinetic aspects of metaphase blockage and recovery. The results indicated that under certain conditions the blocking effects of the drug were reversed; a functional bipolar spindle reorganized, and normal division resulted. Individual HeLa cells were followed by time-lapse cinemicrography prior to, during, and after Colcemid treatment. There was no apparent effect on cells in interphase. Cells entered mitosis at a normal rate and passed through prophase. A spindle was formed in most cells, albeit deformed, stunted, or shrunken. Within a certain range of drug concentrations, the spindle lengths showed characteristic unimodal distributions. After a 2-hr exposure to the drug followed by 1 hr in fresh medium, spindle lengths were restored to normal. Cells arrested in metaphase for periods as long as 5 hr were capable of reconstituting a normal functional spindle. Cells blocked for periods longer than 5 to 6 hr failed to recover.

OBSERVATIONS ON VEGETATIVE NUCLEAR DIVISION IN SACCHAROMYCES CEREVISIAE

In Giemsa-stained preparations of actively growing yeast, nuclear division normally occurred in the isthmus of each bud, but occasionally the nucleus divided in the parent cell and one of the products of division entered the bud, or else division occurred in the bud, following which one of the two nuclei migrated back into the parent cell. The frequency of intrabud divisions was increased by thymine, dihydrothymine, and thymidine. During normal division, the nuclear material entering the bud tended to be organized into two parallel rows, each comprising three densely staining bodies, and the latter were grouped into apparently homologous pairs.