Cell Size Control Research Articles

Big enough for two

Study identifies a signaling pathway that may control cell size by linking membrane transport to mitotic entry.

A link between mitotic entry and membrane growth suggests a novel model for cell size control

Addition of new membrane to the cell surface by membrane trafficking is necessary for cell growth. In this paper, we report that blocking membrane traffic causes a mitotic checkpoint arrest via Wee1-dependent inhibitory phosphorylation of Cdk1. Checkpoint signals are relayed by the Rho1 GTPase, protein kinase C (Pkc1), and a specific form of protein phosphatase 2A (PP2A(Cdc55)). Signaling via this pathway is dependent on membrane traffic and appears to increase gradually during polar bud growth. We hypothesize that delivery of vesicles to the site of bud growth generates a signal that is proportional to the extent of polarized membrane growth and that the strength of the signal is read by downstream components to determine when sufficient growth has occurred for initiation of mitosis. Growth-dependent signaling could explain how membrane growth is integrated with cell cycle progression. It could also control both cell size and morphogenesis, thereby reconciling divergent models for mitotic checkpoint function.

New therapies for tuber-less sclerosis

Tuberous sclerosis complex (TSC) is well known to neurologists because of the high prevalence of epilepsy, autism spectrum disorders, and intellectual disabilities seen in individuals with this genetic disease.1 TSC has been documented in the medical literature for more than 100 years, with clear descriptions of cortical malformations (tubers), skin lesions, epilepsy, and developmental delay. Our understanding of the genetics, cellular signaling pathways, and pathogenesis of TSC has increased dramatically over the past decade. A key discovery was that the proteins encoded by the TSC1 and TSC2 genes normally function to inhibit the mammalian target of rapamycin (mTOR) serine/threonine kinase.2 This was a seminal breakthrough because mTOR kinase activity normally controls cell size, cell proliferation, and differentiation. Because all of these processes are abnormal in TSC, increased mTOR activity was implicated in the pathogenesis of TSC. The mTOR …

The critical size is set at a single-cell level by growth rate to attain homeostasis and adaptation

Budding yeast cells are assumed to trigger Start and enter the cell cycle only after they attain a critical size set by external conditions. However, arguing against deterministic models of cell size control, cell volume at Start displays great individual variability even under constant conditions. Here we show that cell size at Start is robustly set at a single-cell level by the volume growth rate in G1, which explains the observed variability. We find that this growth-rate-dependent sizer is intimately hardwired into the Start network and the Ydj1 chaperone is key for setting cell size as a function of the individual growth rate. Mathematical modelling and experimental data indicate that a growth-rate-dependent sizer is sufficient to ensure size homeostasis and, as a remarkable advantage over a rigid sizer mechanism, it reduces noise in G1 length and provides an immediate solution for size adaptation to external conditions at a population level.

Acetylation of sphingosine kinase 1 regulates cell growth and cell-cycle progression

Sphingosine kinase 1 (SPK1) is a key enzyme in the sphingolipid metabolic pathway. It forms an essential checkpoint to regulate the relative levels of bioactive sphingolipid metabolites, ceramide, sphingosine, and sphingosine 1-phosphate (S1P). Here, we present evidence that SPK1 is acetylated by the intrinsic acetyltransferase activity of p300/cAMP-response element-binding protein (CREB)-binding protein (CBP) at a conserved acetylation motif (the GK motif). This post-translational modification may be an important regulator of SPK1 protein, as acetylation by p300 or CBP increased its stability. Mutation of two lysine (K) residues in its GK motif to either arginine (R) or glutamine (Q) blocked SPK1 ubiquitination and prevented its degradation by the proteasome. The processes of acetylation and ubiquitination may compete for the same lysine residues and, therefore, form a switch for SPK1 protein regulation. Intriguingly, human embryonic kidney (HEK) 293 cells stably expressed the mutated form of SPK1, in which the K residue was mutated to Q (Q-SPK1), and this mutated form mimicked acetylated SPK1. These cells were larger in size and had a slower growth rate compared to cells that expressed wild-type SPK1 (W-SPK1) or the K/R-mutated SPK1 (R-SPK1). These data suggest that SPK1 acetylation plays a key role in cell growth, cell size, and cell-cycle control.

Myopathy caused by mammalian target of rapamycin complex 1 (mTORC1) inactivation is not reversed by restoring mitochondrial function

Mammalian target of rapamycin complex 1 (mTORC1) is central to the control of cell, organ, and body size. Skeletal muscle-specific inactivation of mTORC1 in mice results in smaller muscle fibers, fewer mitochondria, increased glycogen stores, and a progressive myopathy that causes premature death. In mTORC1-deficient muscles, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), which regulates mitochondrial biogenesis and glucose homeostasis, is strongly down-regulated. Here we tested whether induction of mitochondrial biogenesis pharmacologically or by the overexpression of PGC-1α is sufficient to reverse the phenotype of mice deficient for mTORC1. We show that both approaches normalize mitochondrial function, such as oxidative capacity and expression of mitochondrial genes. However, they do not prevent or delay the progressive myopathy. In addition, we find that mTORC1 has a much stronger effect than PGC-1α on the glycogen content in muscle. This effect is based on the strong activation of PKB/Akt in mTORC1-deficient mice. We also show that activation of PKB/Akt not only affects glycogen synthesis but also diminishes glycogen degradation. Thus, our work provides strong functional evidence that mitochondrial dysfunction in mice with inactivated mTORC1 signaling is caused by the down-regulation of PGC-1α. However, our data also show that the impairment of mitochondria does not lead directly to the lethal myopathy.

The reverse, but coordinated, roles of Tor2 (TORC1) and Tor1 (TORC2) kinases for growth, cell cycle and separase-mediated mitosis inSchizosaccharomyces pombe

Target of rapamycin complexes (TORCs), which are vital for nutrient utilization, contain a catalytic subunit with the phosphatidyl inositol kinase-related kinase (PIKK) motif. TORC1 is required for cell growth, while the functions of TORC2 are less well understood. We show here that the fission yeast Schizosaccharomyces pombe TORC2 has a cell cycle role through determining the proper timing of Cdc2 Tyr15 dephosphorylation and the cell size under limited glucose, whereas TORC1 restrains mitosis and opposes securin–separase, which are essential for chromosome segregation. These results were obtained using the previously isolated TORC1 mutant tor2-L2048S in the phosphatidyl inositol kinase (PIK) domain and a new TORC2 mutant tor1-L2045D, which harbours a mutation in the same site. While mutated TORC1 and TORC2 displayed diminished kinase activity and FKBP12/Fkh1-dependent rapamycin sensitivity, their phenotypes were nearly opposite in mitosis. Premature mitosis and the G2–M delay occurred in TORC1 and TORC2 mutants, respectively. Surprisingly, separase/cut1—securin/cut2 mutants were rescued by TORC1/tor2-L2048S mutation or rapamycin addition or even Fkh1 deletion, whereas these mutants showed synthetic defect with TORC2/tor1-L2045D. TORC1 and TORC2 coordinate growth, mitosis and cell size control, such as Wee1 and Cdc25 do for the entry into mitosis.

A Design-of-Experiment Study on the Microcellular Extrusion of Sub-critical CO2 Saturated PLA Pellets

Abstract In this study we explore the use of solid-state nucleation in pellets as a means to decouple cell nucleation from cell growth in extrusion. This is achieved by feeding the extruder with gas-saturated PLA pellets. Cell nucleation occurs, similar to that of the well-studied solid-state batch foaming process, when gas-saturated pellets are crushed and heated in the feed section of the extruder. The polymer melt consequently contains the nucleated cells. Further, the gas content in the pellets provides a means to deliver a controlled amount of blowing agent for cell growth at the die exit. It was found that gas concentrations in partially desorbed pellets offers greater control of part density and foam morphology compared to that of fully saturated pellets at 2 MPa. Using Design-of-Experiment statistical methods it was found that gas concentration in the polymer was the most significant variable influencing foam density, whereas the extrudate cooling method was found to control cell size.

A New Link in the Chain from Amino Acids to mTORC1 Activation

A recent study reveals that the scaffold protein p62 plays a role in linking nutritional cues (amino acids) to the activation of mammalian target of rapamycin complex 1 (mTORC1), a protein kinase that controls cell size and proliferation.

C-di-AMP Is a New Second Messenger in Staphylococcus aureus with a Role in Controlling Cell Size and Envelope Stress

The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP (GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has diadenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13–22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.

The tumor suppressor Tsc1 enforces quiescence of naive T cells to promote immune homeostasis and function

The mechanisms that regulate T cell quiescence are poorly understood. We report that tuberous sclerosis complex 1 (Tsc1) establishes a quiescence program in naive T cells by controlling cell size, cell cycle entry, and responses to T cell receptor stimulation. Loss of quiescence predisposed Tsc1-deficient T cells to apoptosis that resulted in loss of conventional T cells and invariant natural killer T cells. Loss of Tsc1 function dampened in vivo immune responses to bacterial infection. Tsc1-deficient T cells exhibited increased mTORC1 but diminished mTORC2 activities, with mTORC1 activation essential for the disruption of immune homeostasis. Therefore, Tsc1-dependent control of mTOR is crucial in establishing naive T cell quiescence to facilitate adaptive immune function.

Mechanistic Analysis of a DNA Damage-Induced, PTEN-Dependent Size Checkpoint in Human Cells

Following DNA damage, human cells undergo arrests in the G(1) and G(2) phases of the cell cycle and a simultaneous arrest in cell size. We previously demonstrated that the cell size arrest can be uncoupled from the cell cycle arrest by mutational inactivation of the PTEN tumor suppressor gene. Here we show that the cell size checkpoint is inducible by DNA-damaging chemotherapeutic agents as well as by ionizing radiation and is effectively regulated by PTEN but not by its oncogenic counterpart, PIK3CA. Mutational analysis of PTEN and pharmacological inhibition of Akt revealed that modulation of Akt phosphorylation is unnecessary for cell size checkpoint control. To discover putative PTEN regulators and/or effectors involved in size checkpoint control, we employed a novel endogenous epitope tagging (EET) approach, which revealed that endogenous PTEN interacts at the membrane with an actin-remodeling complex that includes actin, gelsolin, and EPLIN. Pharmacological inhibition of actin remodeling in PTEN(+/+) cells recapitulated the lack of size checkpoint control seen in PTEN(-/-) cells. Taken together, these results provide further support for the existence of a DNA damage-inducible size checkpoint that is regulated by a major tumor suppressor, and they provide a novel Akt-independent mechanism by which PTEN controls cell size.

Micro and nano cellular amorphous polymers (PMMA, PS) in supercritical CO 2 assisted by nanostructured CO 2-philic block copolymers – One step foaming process

Microcellular foaming of commodity amorphous polymers, poly(methyl methacrylate) (PMMA), and poly(styrene) (PS) was studied in supercritical CO 2 via a batch one-step process in the presence of block copolymers able to change their foaming behaviour and therefore the porous structures. Triblock (styrene-co-butadiene-co-methylmethacrylate SBM, methylmethacrylate-co-butylacrylate-co-methylmethacrylate MAM) terpolymers were blended to PS or PMMA by extrusion. They showed advantages compared to classical PS-PMMA polymer blends in terms of cell size control and reduction of cell size. Foaming is carried out on bulk injection molded samples which were saturated under high pressures of CO 2 (300 bars) at different temperatures (25° C to 80 °C) and different depressurization rates (pressure drop rates from 150 bar/min to 12 bar/min). Very distinct cellular structures and densities were controlled by varying either the copolymer type or the foaming conditions (T,P). Cell sizes ranged from 0.2 μm to 200 μm, and densities from 0.30 g/cm 3 to 1 g/cm 3 in the polymers considered. Particularly, when triblock copolymers were able to self organize (nanostructuring) in a polymer matrix, they became phase separated at a nanometer level, presenting nanostructured polymers matrixes. To conclude the study, a possible nanostructuring mechanism is suggested based on the interplay between rubbery and highly CO 2-philic blocks/rigid and less CO 2-philic blocks. It is demonstrated that block copolymer additives are a good pathway towards micro and ultra microcellular supercritical CO 2 foaming of amorphous polymers.

Rac1 Regulates the Activity of mTORC1 and mTORC2 and Controls Cellular Size

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that exists in two separate complexes, mTORC1 and mTORC2, that function to control cell size and growth in response to growth factors, nutrients, and cellular energy levels. Low molecular weight GTP-binding proteins of the Rheb and Rag families are key regulators of the mTORC1 complex, but regulation of mTORC2 is poorly understood. Here, we report that Rac1, a member of the Rho family of GTPases, is a critical regulator of both mTORC1 and mTORC2 in response to growth-factor stimulation. Deletion of Rac1 in primary cells using an inducible-Cre/Lox approach inhibits basal and growth-factor activation of both mTORC1 and mTORC2. Rac1 appears to bind directly to mTOR and to mediate mTORC1 and mTORC2 localization at specific membranes. Binding of Rac1 to mTOR does not depend on the GTP-bound state of Rac1, but on the integrity of its C-terminal domain. This function of Rac1 provides a means to regulate mTORC1 and mTORC2 simultaneously.

Elucidating the functional role of endoreduplication in tomato fruit development

Endoreduplication is the major source of endopolyploidy in higher plants. The process of endoreduplication results from the ability of cells to modify their classical cell cycle into a partial cell cycle where DNA synthesis occurs independently from mitosis. Despite the ubiquitous occurrence of the phenomenon in eukaryotic cells, the physiological meaning of endoreduplication remains vague, although several roles during plant development have been proposed, mostly related to cell differentiation and cell size determination. Here recent advances in the knowledge of endoreduplication and fruit organogenesis are reviewed, focusing on tomato (Solanum lycopersicum) as a model, and the functional analyses of endoreduplication-associated regulatory genes in tomato fruit are described. The cyclin-dependent kinase inhibitory kinase WEE1 and the anaphase promoting complex activator CCS52A both participate in the control of cell size and the endoreduplication process driving cell expansion during early fruit development in tomato. Moreover the fruit-specific functional analysis of the tomato CDK inhibitor KRP1 reveals that cell size and fruit size determination can be uncoupled from DNA ploidy levels, indicating that endoreduplication acts rather as a limiting factor for cell growth. The overall functional data contribute to unravelling the physiological role of endoreduplication in growth induction of fleshy fruits.

Primary cilia regulate mTORC1 activity and cell size through Lkb1

The mTOR pathway is the central regulator of cell size. External signals from growth factors and nutrients converge on the mTORC1 multi-protein complex to modulate downstream targets, but how the different inputs are integrated and translated into specific cellular responses is incompletely understood. Deregulation of the mTOR pathway occurs in polycystic kidney disease (PKD), where cilia (filiform sensory organelles) fail to sense urine flow because of inherited mutations in ciliary proteins. We therefore investigated if cilia have a role in mTOR regulation. Here, we show that ablation of cilia in transgenic mice results in enlarged cells when compared with control animals. In vitro analysis demonstrated that bending of the cilia by flow is required for mTOR downregulation and cell-size control. Surprisingly, regulation of cell size by cilia is independent of flow-induced calcium transients, or Akt. However, the tumour-suppressor protein Lkb1 localises in the cilium, and flow results in increased AMPK phosphorylation at the basal body. Conversely, knockdown of Lkb1 prevents normal cell-size regulation under flow conditions. Our results demonstrate that the cilium regulates mTOR signalling and cell size, and identify the cilium-basal body compartment as a spatially restricted activation site for Lkb1 signalling.

Production, cellular structure and thermal conductivity of microcellular (methyl methacrylate)–(butyl acrylate)–(methyl methacrylate) triblock copolymers

AbstractMicrocellular foaming of a (methyl methacrylate)–(butyl acrylate)–(methyl methacrylate) triblock copolymer was carried out by means of supercritical CO2 in a single‐step process. The experiments were performed at 40 °C using a pressure of 300 bar (30 MPa) during 24 h. The depressurization times were modified from 2 to 30 min, leading to cell sizes from 10 to 100 µm, and relative densities from 0.11 to 0.17. It was found that the key parameter to control cell size and density was depressurization time: longer depressurization times generated larger cell sizes and lower densities. The thermal conductivity of these materials was measured using the transient plane source technique, and it was found that this decreased as the density was reduced. Various models for the prediction of thermal conductivity by conduction were tested. It was found that all the models underestimated the experimental results due to a significant contribution of radiation heat flow for these materials. Copyright © 2010 Society of Chemical Industry

Sirolimus attenuates disease progression in an orthologous mouse model of human autosomal dominant polycystic kidney disease

In autosomal dominant polycystic kidney disease (ADPKD), abnormal proliferation of tubular cells drives cyst development and growth. Sirolimus, an inhibitor of the protein kinase mammalian target of rapamycin (mTOR) and a potent anti-proliferative agent, decreases cyst growth in several genetically distinct rodent models of polycystic kidney disease (PKD). We determined here the effect of sirolimus on renal cyst growth in Pkd2WS25/- mice; an ortholog of human ADPKD involving mutation of the Pkd2 gene. In Pkd2WS25/- mice treated with sirolimus, both the two kidney/total body weight (2K/TBW) ratio and the cyst volume density (CVD) were significantly decreased by over half compared with untreated mice suffering with PKD. However, there was no effect on the increased blood urea nitrogen (BUN) levels as an index of kidney function. There are two distinct complexes containing mTOR depending on its binding partners: mTORC1 and mTORC2. Western blot analysis of whole kidney lysates and immunohistochemistry of the cysts found that phospho-S6 ribosomal protein, a marker of mTORC1 activity, was increased in Pkd2WS25/- mice and its phosphorylation was decreased by sirolimus treatment. Phospho-Akt at serine 473, a marker associated with mTORC2 activity, was not different between Pkd2WS25/- mice and normal littermate controls. Hence, our study found that inhibition of mTORC1 by sirolimus correlated with decreased renal cyst growth in this model of human ADPKD but had no effect on the decline in renal function.

How does the plant proteasome control leaf size?

The ubiquitin/26S proteasome pathway plays a central role in the degradation of short-lived regulatory proteins to control many cellular events. The Arabidopsis genome contains two genes, AtRPT2a and AtRPT2b, which encode paralog molecules of the RPT2 subunit of 19S proteasome. We demonstrated that mutation of the AtRPT2a gene causes a specific phenotype of enlarged leaves due to increased cell size in correlation with expanded endoreduplication. This phenotype was also observed in the knockout mutant of AtRPT5a, which encodes one of the paralogs of the RPT5 subunit. Taken together, this suggests that a cell size-specific proteasome consisting of AtRPT2a and AtRPT5a is involved in controlling cell size during leaf development.

Microcellular foaming of polymethylmethacrylate in a batch supercritical CO2 process: Effect of microstructure on compression behavior

AbstractMicrocellular foaming of reinforced core/shell Polymethylmethacrylate (PMMA) was carried out by means of supercritical CO2 in a single‐step process. Samples were produced using a technique based on the saturation of the polymer under high pressure of CO2 (300 bars, 40°C), and cellular structure was controlled by varying the depressurization rate from 0.5 bar/s to 1.6 × 10−2 bar/s leading to cell sizes from 1 μm to 200 μm, and densities from 0.8 to 1.0 g/cm3. It was found that the key parameter to control cell size was depressurization rate, and larger depressurization rates generated bigger cell sizes. On the other hand, variation of the density of the samples was not so considerable. Low rate compression tests were carried out, analyzing the dependence of mechanical parameters such as elastic modulus, yield stress and densification strain with cell size. Moreover, the calculation of the energy absorbed for each sample is presented, showing an optimum of energy absorption up to 50% of deformation in the micrometer cellular range (here at a cell size of about 5 μm). To conclude, a brief comparison between neat PMMA and the core/shell reinforced PMMA has been carried out, analyzing the effect of the core/shell particles in the foaming behavior and mechanical properties. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010