Growth Of Daughter Cells Research Articles

Plasticity in the cell division processes of obligate intracellular bacteria.

Most bacteria divide through a highly conserved process called binary fission, in which there is symmetric growth of daughter cells and the synthesis of peptidoglycan at the mid-cell to enable cytokinesis. During this process, the parental cell replicates its chromosomal DNA and segregates replicated chromosomes into the daughter cells. The mechanisms that regulate binary fission have been extensively studied in several model organisms, including Eschericia coli, Bacillus subtilis, and Caulobacter crescentus. These analyses have revealed that a multi-protein complex called the divisome forms at the mid-cell to enable peptidoglycan synthesis and septation during division. In addition, rod-shaped bacteria form a multi-protein complex called the elongasome that drives sidewall peptidoglycan synthesis necessary for the maintenance of rod shape and the lengthening of the cell prior to division. In adapting to their intracellular niche, the obligate intracellular bacteria discussed here have eliminated one to several of the divisome gene products essential for binary fission in E. coli. In addition, genes that encode components of the elongasome, which were mostly lost as rod-shaped bacteria evolved into coccoid organisms, have been retained during the reductive evolutionary process that some coccoid obligate intracellular bacteria have undergone. Although the precise molecular mechanisms that regulate the division of obligate intracellular bacteria remain undefined, the studies summarized here indicate that obligate intracellular bacteria exhibit remarkable plasticity in their cell division processes.

Penicillin-binding proteins regulate multiple steps in the polarized cell division process of Chlamydia

Chlamydia trachomatis serovar L2 and Chlamydia muridarum, which do not express FtsZ, undergo polarized cell division. During division, peptidoglycan assembles at the pole of dividing Chlamydia trachomatis cells where daughter cell formation occurs, and peptidoglycan regulates at least two distinct steps in the polarized division of Chlamydia trachomatis and Chlamydia muridarum. Cells treated with inhibitors that prevent peptidoglycan synthesis or peptidoglycan crosslinking by penicillin-binding protein 2 (PBP2) are unable to initiate polarized division, while cells treated with inhibitors that prevent peptidoglycan crosslinking by penicillin-binding protein 3 (PBP3/FtsI) initiate polarized division, but the process arrests at an early stage of daughter cell growth. Consistent with their distinct roles in polarized division, peptidoglycan organization is different in cells treated with PBP2 and PBP3-specific inhibitors. Our analyses indicate that the sequential action of PBP2 and PBP3 drives changes in peptidoglycan organization that are essential for the polarized division of these obligate intracellular bacteria. Furthermore, the roles we have characterized for PBP2 and PBP3 in regulating specific steps in chlamydial cell division have not been described in other bacteria.

Mammalian cell growth dynamics in mitosis.

The extent and dynamics of animal cell biomass accumulation during mitosis are unknown, primarily because growth has not been quantified with sufficient precision and temporal resolution. Using the suspended microchannel resonator and protein synthesis assays, we quantify mass accumulation and translation rates between mitotic stages on a single-cell level. For various animal cell types, growth rates in prophase are commensurate with or higher than interphase growth rates. Growth is only stopped as cells approach metaphase-to-anaphase transition and growth resumes in late cytokinesis. Mitotic arrests stop growth independently of arresting mechanism. For mouse lymphoblast cells, growth in prophase is promoted by CDK1 through increased phosphorylation of 4E-BP1 and cap-dependent protein synthesis. Inhibition of CDK1-driven mitotic translation reduces daughter cell growth. Overall, our measurements counter the traditional dogma that growth during mitosis is negligible and provide insight into antimitotic cancer chemotherapies.

Tropomyosin and Profilin Cooperate to Promote Formin-Mediated Actin Nucleation and Drive Yeast Actin Cable Assembly

Tropomyosins comprise a large family of actin-binding proteins with critical roles in diverse actin-basedprocesses [1], but our understanding of how theymechanistically contribute to actin filament dynamics has been limited. We addressed this question in S.cerevisiae, where tropomyosins (Tpm1 and Tpm2), profilin (Pfy1), and formins (Bni1 and Bnr1) are required for the assembly of an array of actin cables that facilitate polarized vesicle delivery and daughter cell growth. Formins drive cable formation by promoting actin nucleation and by accelerating actin filament elongation together with profilin [2]. In contrast, how tropomyosins contribute mechanistically to cable formation has been unclear, but genetic studies demonstrate that Tpm1 plays a more important role than Tpm2 [3, 4]. Here, we found thatloss of TPM1 in strains lacking BNR1, but not BNI1, leads to severe defects in cable formation, polarized secretion, and cell growth, suggesting that TPM1 function is required for proper Bni1-mediated cable assembly. Furthermore, invitro total internal reflection fluorescence (TIRF) microscopy demonstratedthat Tpm1 strongly enhances Bni1-mediated, butnot Bnr1-mediated, actin nucleation without affecting filament elongation rate, whereas Tpm2 has no effects on Bni1 or Bnr1. Tpm1 stimulation of Bni1-mediated nucleation also requires profilin and its interactions with both G-actin and formins. Together, these results demonstrate that yeast Tpm1 works in concert with profilin to promote formin-dependent nucleation of actin cables, thus expanding our understanding of how specific tropomyosin isoforms influence actin dynamics.

Unidirectional P-body transport during the yeast cell cycle.

P-bodies belong to a large family of RNA granules that are associated with post-transcriptional gene regulation, conserved from yeast to mammals, and influence biological processes ranging from germ cell development to neuronal plasticity. RNA granules can also transport RNAs to specific locations. Germ granules transport maternal RNAs to the embryo, and neuronal granules transport RNAs long distances to the synaptic dendrites. Here we combine microfluidic-based fluorescent microscopy of single cells and automated image analysis to follow p-body dynamics during cell division in yeast. Our results demonstrate that these highly dynamic granules undergo a unidirectional transport from the mother to the daughter cell during mitosis as well as a constrained “hovering” near the bud site half an hour before the bud is observable. Both behaviors are dependent on the Myo4p/She2p RNA transport machinery. Furthermore, single cell analysis of cell size suggests that PBs play an important role in daughter cell growth under nutrient limiting conditions.

A Rab8 Guanine Nucleotide Exchange Factor-Effector Interaction Network Regulates Primary Ciliogenesis

Primary cilia are microtubule-based solitary membrane projections on the cell surface that play important roles in signaling and development. Recent studies have demonstrated that polarized vesicular trafficking involving the small GTPase Rab8 and its guanine nucleotide exchange factor Rabin8 is essential for primary ciliogenesis. In this study, we show that a highly conserved region of Rabin8 is pivotal for its activation as a guanine nucleotide exchange factor for Rab8. In addition, in its activated conformation, Rabin8 interacts with Sec15, a subunit of the exocyst and downstream effector of Rab8. Expression of constitutively activated Rab8 promotes the association of Sec15 with Rabin8. Using immunofluorescence microscopy, we found that Sec15 co-localized with Rab8 along the primary cilium. Inhibition of Sec15 function in cells led to defects in primary ciliogenesis. The Rabin8-Rab8-Sec15 interaction may couple the activation of Rab8 to the recruitment of the Rab8 effector and is involved in the regulation of vesicular trafficking for primary cilium formation.

How to grow a bud: an importin acts in asymmetric division

The growth of daughter cells in budding yeast is a classic model for investigating mechanisms involved in asymmetric cell division. An unexpected collaboration between the DEAD-box protein Dbp5 and the nuclear transport receptor Kap104 controls localized protein synthesis at the bud tip during mitosis.

Regulation of Platelet Dense Granule Secretion by the Ral GTPase-Exocyst Pathway

Non-hydrolyzable GTP analogues, such as guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp), induce granule secretion from permeabilized platelets in the absence of increased intracellular Ca(2+). Here, we show that the GppNHp-induced dense granule secretion from permeabilized platelets occurred concomitantly with the activation of small GTPase Ral. This secretion was inhibited by the addition of GTP-Ral-binding domain (RBD) of Sec5, which is a component of the exocyst complex known to function as a tethering factor at the plasma membrane for vesicles. We generated an antibody against Sec5-RBD, which abolished the interaction between GTP-Ral and the exocyst complex in vitro. The addition of this antibody inhibited the GppNHp-induced secretion. These data indicate that Ral mediates the GppNHp-induced dense granule secretion from permeabilized platelets through interaction with its effector, the exocyst complex. Furthermore, GppNHp enhanced the Ca(2+) sensitivity of dense granule secretion from permeabilized platelets, and this enhancement was inhibited by Sec5-RBD. In intact platelets, the association between Ral and the exocyst complex was induced by thrombin stimulation with a time course similar to that of dense granule secretion and Ral activation. Taken together, our results suggest that the Ral-exocyst pathway participates in the regulation of platelet dense granule secretion by enhancing the Ca(2+) sensitivity of the secretion.

Exo70p mediates the secretion of specific exocytic vesicles at early stages of the cell cycle for polarized cell growth

In budding yeast, two classes of post-Golgi secretory vesicles carrying different sets of cargoes typified by Bgl2p and invertase are delivered to the plasma membrane for secretion. The exocyst is implicated in tethering these vesicles to the daughter cell membrane for exocytosis. In this study, we report that mutations in the exocyst component Exo70p predominantly block secretion of the Bgl2p vesicles. Furthermore, a defect in invertase vesicle trafficking caused by vps1Δ or pep12Δ in the exo70 mutant background is detrimental to the cell. The secretion defect in exo70 mutants was most pronounced during the early budding stage, which affected daughter cell growth. The selective secretion block does not occur at the vesicle formation or sorting stage because the exocytic vesicles are properly generated and protein processing is normal in the exo70 mutants. Our study suggests that Exo70p functions primarily at early stages of the cell cycle in Bgl2p vesicle secretion, which is critical for polarized cell growth.

Hyphal Guidance and Invasive Growth in Candida albicans Require the Ras-Like GTPase Rsr1p and Its GTPase-Activating Protein Bud2p

Candida albicans, the most prevalent fungal pathogen of humans, causes superficial mycoses, invasive mucosal infections, and disseminated systemic disease. Many studies have shown an intriguing association between C. albicans morphogenesis and the pathogenesis process. For example, hyphal cells have been observed to penetrate host epithelial cells at sites of wounds and between cell junctions. Ras- and Rho-type GTPases regulate many morphogenetic processes in eukaryotes, including polarity establishment, cell proliferation, and directed growth in response to extracellular stimuli. We found that the C. albicans Ras-like GTPase Rsr1p and its predicted GTPase-activating protein Bud2p localized to the cell cortex, at sites of incipient daughter cell growth, and provided landmarks for the positioning of daughter yeast cells and hyphal cell branches, similar to the paradigm in the model yeast Saccharomyces cerevisiae. However, in contrast to S. cerevisiae, CaRsr1p and CaBud2p were important for morphogenesis: C. albicans strains lacking Rsr1p or Bud2p had abnormal yeast and hyphal cell shapes and frequent bends and promiscuous branching along the hypha and were unable to invade agar. These defects were associated with abnormal actin patch polarization, unstable polarisome localization at hyphal tips, and mislocalized septin rings, consistent with the idea that GTP cycling of Rsr1p stabilizes the axis of polarity primarily to a single focus, thus ensuring normal cell shape and a focused direction of polarized growth. We conclude that the Rsr1p GTPase functions as a polarity landmark for hyphal guidance and may be an important mediator of extracellular signals during processes such as host invasion.

Involvement of DivIVA in the morphology of the rod-shaped actinomycete Brevibacterium lactofermentum.

In Brevibacterium lactofermentum, as in many Gram-positive bacteria, a divIVA gene is located downstream from the dcw cluster of cell-division- and cell-wall-related genes. This gene (divIVA(BL)) is mostly expressed during exponential growth, and the protein encoded, DivIVA(BL,) bears some sequence similarity to antigen 84 (Ag84) from mycobacteria and was detected with monoclonal antibodies against Ag84. Disruption experiments using an internal fragment of the divIVA(BL) gene or a disrupted divIVA(BL) cloned in a suicide conjugative plasmid were unsuccessful, suggesting that the divIVA(BL) gene is needed for cell viability in BREV: lactofermentum. Transformation of BREV: lactofermentum with a multicopy plasmid containing divIVA(BL) drastically altered the morphology of the corynebacterial cells, which became larger and bulkier, and a GFP fusion to DivIVA(BL) mainly localized to the ends of corynebacterial cells. This localization pattern, together with the overproduction phenotype, suggests that DivIVA may be important in regulating the apical growth of daughter cells.

Genetic models in applied physiology: invited review: effect of oxygen deprivation on cell cycle activity: a profile of delay and arrest.

One of the most fascinating fields that have emanated in the past few decades is developmental biology. This is not only the case from a research point of view but also from the angle of clinical care and treatment strategies. It is now well demonstrated that there are many diseases (some believe all diseases) that have their roots in embryogenesis or in early life, where nature and environment often team up to facilitate the genesis of disease. There is probably no better example to illustrate the interactions between nature and environment than in early life, as early as in the first several cell cycles. As will be apparent in this review, the cell cycle is a very regulated activity and this regulation is genetic in nature, with checkpoint proteins playing an important role in controlling the timing, the size, and the growth of daughter cells. However, it is also very clear, as will be discussed in this work, that the microenvironment of the first dividing cells is so important for the outcome of the organism. In this review, we will focus on the effect of one stress, that of hypoxia, on the young embryo and its cell division and growth. We will first review some of the cell cycle definitions and stages and then review briefly our current knowledge and its gaps in this area.

Cell cycle-dependent assembly of a Gin4-septin complex.

Gin4, a Nim1-related kinase, is required in budding yeast for localization of the septins and for proper control of daughter cell growth during G2/M. Gin4 becomes hyperphosphorylated when cells enter mitosis, leading to activation of Gin4 kinase activity. In this study, we have used immunoaffinity chromatography to identify proteins that associate with Gin4 during mitosis, with the goal of finding targets of Gin4 kinase activity and proteins that play a role in Gin4 activation. We show that during mitosis Gin4 is assembled into a multiprotein complex that includes Nap1, Bni5, the septins, and at least two molecules of Gin4. The associated Gin4 molecules present in this complex phosphorylate each other, leading to Gin4 hyperphosphorylation. Furthermore, the Shs1 septin present in the complex undergoes Gin4-dependent phosphorylation during mitosis and appears to be a substrate of Gin4 in vitro, suggesting that it is a target of Gin4 kinase activity in vivo. Genetic data support the idea that Shs1 is an important target of Gin4 kinase activity. Association of Gin4 with the septins during mitosis requires Shs1, Nap1, Cla4, Elm1, and the kinase activities of Gin4 and Cdc28. Self-association of Gin4 molecules requires Shs1 but not Cla4 or Nap1. Previous work has suggested that the septins function together as a tight complex, and we found that the majority of the Shs1 in the cell is tightly bound to the other septins Cdc3, Cdc10, Cdc11, and Cdc12. Interestingly, however, Shs1 can bind to Gin4 and induce Gin4 oligomerization under conditions in which the Cdc11 septin does not bind to Gin4, suggesting that Shs1 can function independently of the other septins. Taken together, these findings suggest that highly regulated protein-binding events ensure that the Gin4 kinase is activated only during mitosis and only in association with Shs1, a likely in vivo substrate of Gin4. In addition, these results provide clues to how Gin4 may regulate the localization or function of the septins.

Volume growth of daughter and parent cells during the cell cycle of Saccharomyces cerevisiae a/alpha as determined by image cytometry.

The pattern of volume growth of Saccharomyces cerevisiae a/alpha was determined by image cytometry for daughter cells and consecutive cycles of parent cells. An image analysis program was specially developed to measure separately the volume of bud and mother cell parts and to quantify the number of bud scars on each parent cell. All volumetric data and cell attributes (budding state, number of scars) were stored in such a way that separate volume distributions of cells or cell parts with any combination of properties--for instance, buds present on mothers with two scars or cells without scars (i.e., daughter cells) and without buds--could be obtained. By a new method called intersection analysis, the average volumes of daughter and parent cells at birth and at division could be determined for a steady-state population. These volumes compared well with those directly measured from cells synchronized by centrifugal elutriation. During synchronous growth of daughter cells, the pattern of volume increase appeared to be largely exponential. However, after bud emergence, larger volumes than those predicted by a continuous exponential increase were obtained, which confirms the reported decrease in buoyant density. The cycle times calculated from the steady-state population by applying the age distribution equation deviated from those directly obtained from the synchronized culture, probably because of inadequate scoring of bud scars. Therefore, for the construction of a volume-time diagram, we used volume measurements obtained from the steady-state population and cycle times obtained from the synchronized population. The diagram shows that after bud emergence, mother cell parts continue to grow at a smaller rate, increasing about 10% in volume during the budding period. Second-generation daughter cells, ie., cells born from parents left with two scars, were significantly smaller than first-generation daughter cells. Second- and third-generation parent cells showed a decreased volume growth rate and a shorter budding period than that of daughter cells.

STUDIES ON BACTERIAL CELL MULTIPLICATION: SYNCHRONOUS SEGMENTATION OF FILAMENTOUS CELLS OF LACTOBACILLUS DELBRUECKII

Several nutritional conditions were tested for their capacity to synchronize the segmentation of filamentous cells of Lactobacillus delbrueckii produced by growth under vitamin B12 deficiency. A medium containing adenine, guanine, uracil, vitamin B2, B12, folic acid, niacin, and pantothenic acid, beside glucose, amino acid, and inorganic salts was found to be most effective in synchronizing the segmentation of filamentous cells which was not followed by separation and growth of daughter cells. Cytological observations disclosed that the segmentation of filamentous cells in this medium was preceded by an increase in stainability with HCl-Giemsa and the rearrangement in cells of nuclear bodies.

The effects of indoleacetic acid and kinetin on the growth of some microorganisms

The growth rates of A. proteus, E. coli bacteria, and two species of yeast are changed by the presence of kinetin and/or the plant auxin, indoleacetic acid.The growth rate of A. proteus is increased by the presence of kinetin. Indoleacetic acid has no apparent effect on this cell.Kinetin increases the growth rate of E. coli in a salt-glucose medium. Adenine also accelerates growth. However, isotopic competition experiments indicated that the two purines do not compete; they must be incorporated into different compounds.IAA depresses the population growth rate of the yeasts, S. cerevisiae and S. pombe. This decrease can be accounted for entirely as a change in the interphase growth rate of the individual cell.Kinetin, by itself, has no apparent effect on yeast growth. In the presence of IAA, kinetin, over a wide concentration range, greatly slows culture growth for several generations. The growth rate progressively approaches and eventually exceeds the control rate. Single cells of the early log population grow at the same rate as those in medium containing only IAA but either are completely blocked from dividing or take longer to divide.The yeast cells are changed physiologically by kinetin. When washed and resuspended in control medium, their growth rate exceeds the control rate for three or four generations. Also, if inhibited cells from late log phase are resuspended in fresh medium containing IAA-plus-kinetin, they grow at a rate faster than the controls even in the presence of the chemicals. Although these fast-growing cells have a normal growth rate during interphase, they also continue to enlarge during the division period. This enlargement during division is caused by the premature initiation of daughter cell growth (before the completion of cytokinesis); in the inhibited cells the start of interphase is delayed.In yeast, it was shown that kinetin does not serve as a source of purine, since it did not support growth of eight adenine-requiring mutants.The observed increase or decrease in the time between mitosis and daughter-cell growth of yeast may be ascribed to an action by kinetin on a growth regulating system which is duplicated at this stage of the cell cycle.