Cell Size Control Research Articles

Coordinated maintenance of muscle cell size control by AMP‐activated protein kinase

Skeletal muscle mass is regulated by signaling pathways that govern protein synthesis and cell proliferation, and the mammalian target of rapamycin (mTOR) plays a key role in these processes. Recent studies suggested the crucial role of AMP-activated protein kinase (AMPK) in the inhibition of protein synthesis and cell growth. Here, we address the role of AMPK in the regulation of muscle cell size in vitro and in vivo. The size of AMPK-deficient myotubes was 1.5-fold higher than for controls. A marked increase in p70S6K Thr(389) and rpS6 Ser-235/236 phosphorylation was observed concomitantly with an up-regulation of protein synthesis rate. Treatment with rapamycin prevented p70S6K phosphorylation and rescued cell size control in AMPK-deficient cells. Importantly, myotubes lacking AMPK were resistant to further cell size increase beyond AMPK deletion alone, as MyrAkt-induced hypertrophy was absent in these cells. Moreover, in skeletal muscle-specific deficient AMPKalpha1/alpha2 KO mice, soleus muscle showed a higher mass with myofibers of larger size and was associated with increased p70S6K and rpS6 phosphorylation. Our results uncover the role of AMPK in the maintenance of muscle cell size control and highlight the crosstalk between AMPK and mTOR/p70S6K signaling pathways coordinating a metabolic checkpoint on cell growth.

Mechanisms Controlling Cell Size and Shape during Isotropic Cell Spreading

Cell motility is important for many developmental and physiological processes. Motility arises from interactions between physical forces at the cell surface membrane and the biochemical reactions that control the actin cytoskeleton. To computationally analyze how these factors interact, we built a three-dimensional stochastic model of the experimentally observed isotropic spreading phase of mammalian fibroblasts. The multiscale model is composed at the microscopic levels of three actin filament remodeling reactions that occur stochastically in space and time, and these reactions are regulated by the membrane forces due to membrane surface resistance (load) and bending energy. The macroscopic output of the model (isotropic spreading of the whole cell) occurs due to the movement of the leading edge, resulting solely from membrane force-constrained biochemical reactions. Numerical simulations indicate that our model qualitatively captures the experimentally observed isotropic cell-spreading behavior. The model predicts that increasing the capping protein concentration will lead to a proportional decrease in the spread radius of the cell. This prediction was experimentally confirmed with the use of Cytochalasin D, which caps growing actin filaments. Similarly, the predicted effect of actin monomer concentration was experimentally verified by using Latrunculin A. Parameter variation analyses indicate that membrane physical forces control cell shape during spreading, whereas the biochemical reactions underlying actin cytoskeleton dynamics control cell size (i.e., the rate of spreading). Thus, during cell spreading, a balance between the biochemical and biophysical properties determines the cell size and shape. These mechanistic insights can provide a format for understanding how force and chemical signals together modulate cellular regulatory networks to control cell motility.

Abstract 1124: Characterization of a PTEN C2 loop mutation in a human breast cancer cell line

Abstract PTEN is a major tumor suppressor gene inactivated in over 30% of human cancer. It encodes a lipid phosphatase with specificity towards phosphatidylinositol (3,4,5) triphosphate (PIP3). PTEN phosphatase activity is crucial to its ability to control cell size, proliferation, motility and survival. The role of PTEN in human breast cancer is well-established. Although the frequency of PTEN mutation in human breast cancer is low (5-10%), a significant fraction of advanced breast cancer has reduced PTEN expression. Furthermore, germ line mutation in PTEN gene constitutes the genetic basis of Cowden's Disease (CD) Syndrome. CD is a harmatoma syndrome with patients displaying aberrant overgrowth of multiple tissue types. Individuals are at a greater risk of developing malignancy of the breast, colon, skin and thyroid. Our laboratory has identified a unique mutation in the C2 loop of PTEN from a human metastatic breast carcinoma cell line, MDA-MB-453. A G to A change substitutes the glutamate (GAG) at codon 307 (E307) to a lysine (AAA). The PTEN C2 loop (aa282-312) is a region on the cytosolic side of the C2 membrane-binding domain. Mono-ubiquitination at K289 within the C2 loop has been shown to promote nuclear translocation. More importantly, unlike the wild-type protein, the E307K mutant is almost undetectable in the nuclear compartment of MDA-MB-453. Biochemically, PTEN E307K mutant displayed similar phosphatase activity towards soluble lipid substrates. More importantly, PTEN E307K displays a striking ability to form an affinity complex with the p53 tumor suppressor. Consistent with this finding, the expression of E307K exerts chemoprotective effect against cisplatinum induced cell death. Based on this preliminary data, we hypothesize that the negatively-charged glutamate residues of PTEN C2 loop play regulatory roles in nuclear translocation and p53-binding. Mutating those residues to lysine creates cryptic ubiquitination sites that subvert the natural ubiquitination at K289, and thus impedes nuclear translocation. Funding: DOD - DAMD17-03-1-0682 (AMC); NCI - RO1CA095063 (AMC), T32CA078207 (GS); Midwest Athletes against Childhood Cancer (AMC); Advancing Healthy Wisconsin Funds (AMC) Acknowledgement: Dr. Xuejun Jiang (Memorial Sloan Kettering Cancer Center, NY) and Dr. Zhen-Qiang Pan (Mount Sinai School of Medicine, NY) for providing the NEDD4-1 and ubiquitination reagents Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1124.

A mathematical model for cell size control in fission yeast

Experimental investigations of cell size control in fission yeast Schizosaccharomyces pombe have illustrated that the cell cycle features ‘sizer’ and ‘timer’ phases which are distinguished by a growth rate changing point. Based on current biological knowledge of fission yeast size control, we propose here a model of ordinary differential equations (ODEs) for a possible explanation of the facts and control mechanism which is coupled with the cell cycle. Simulation results of the ODE model are demonstrated to agree with experimental data for the wild type and the cdc2-33 mutant. We show that the coupling of cell growth to cell division by translational control may account for observed properties of size control in fission yeast. As the translational control in the expression of cycle proteins Cdc13 and Cdc25 constructs positive feedback loops, the dynamical activities of the key components undergoes a rapid rising after a preliminary stage of slow increase. The coupling of this dynamical behavior to the elongation of the cell naturally gives rise to a rate change point and to ‘sizer’ and ‘timer’ phases, which characterize the cell cycle of fission yeast.

The alpha-subunit of the Arabidopsis heterotrimeric G protein, GPA1, is a regulator of transpiration efficiency.

Land plants must balance CO2 assimilation with transpiration in order to minimize drought stress and maximize their reproductive success. The ratio of assimilation to transpiration is called transpiration efficiency (TE). TE is under genetic control, although only one specific gene, ERECTA, has been shown to regulate TE. We have found that the alpha-subunit of the heterotrimeric G protein in Arabidopsis (Arabidopsis thaliana), GPA1, is a regulator of TE. gpa1 mutants, despite having guard cells that are hyposensitive to abscisic acid-induced inhibition of stomatal opening, have increased TE under ample water and drought stress conditions and when treated with exogenous abscisic acid. Leaf-level gas-exchange analysis shows that gpa1 mutants have wild-type assimilation versus internal CO2 concentration responses but exhibit reduced stomatal conductance compared with ecotype Columbia at ambient and below-ambient internal CO2 concentrations. The increased TE and reduced whole leaf stomatal conductance of gpa1 can be primarily attributed to stomatal density, which is reduced in gpa1 mutants. GPA1 regulates stomatal density via the control of epidermal cell size and stomata formation. GPA1 promoter::beta-glucuronidase lines indicate that the GPA1 promoter is active in the stomatal cell lineage, further supporting a function for GPA1 in stomatal development in true leaves.

Fission yeast TORC1 regulates phosphorylation of ribosomal S6 proteins in response to nutrients and its activity is inhibited by rapamycin

Cellular activities are regulated by environmental stimuli through protein phosphorylation. Target of rapamycin (TOR), a serine/threonine kinase, plays pivotal roles in cell proliferation and cell growth in response to nutrient status. In Schizosaccharomyces pombe, TORC1, which contains Tor2, plays crucial roles in nutrient response. Here we find a nitrogen-regulated phosphoprotein, p27, in S. pombe using the phospho-Akt substrate antibody. Response of p27 phosphorylation to nitrogen availability is mediated by TORC1 and the TSC-Rhb1 signaling, but not by TORC2 or other nutrient stress-related pathways. Database and biochemical analyses indicate that p27 is identical to ribosomal protein S6 (Rps6). Ser235 and Ser236 in Rps6 are necessary for Rps6 phosphorylation by TORC1. These Rps6 phosphorylations are dispensable for cell viability. Rps6 phosphorylation by TORC1 also responds to availability of glucose and is inhibited by osmotic and oxidative stresses. Rapamycin inhibits the ability of TORC1 to phosphorylate Rps6, owing to interaction of the rapamycin-FKBP12 complex with the FRB domain in Tor2. Rapamycin also leads to a decrease in cell size in a TORC1-dependent manner. Our findings demonstrate that the nutrient-responsive and rapamycin-sensitive TORC1-S6 signaling exists in S. pombe, and that this pathway plays a role in cell size control.

Theoretical and visual study of bubble dynamics in foam injection molding

AbstractThis article presents an experimental observation and a theoretical prediction of bubble dynamics in foam injection molding process with a main focus on the cell collapse phenomenon under pressure. Using a visualizing setup, cell growth behavior under a nonisothermal condition was monitored. In conjunction with the growth behavior, dynamics of cell collapse under different pressures and the effect of growing time on collapse behavior and final cell size were studied. Theoretical simulation of bubble behavior included power law model, which predicted bubble dynamics during foaming process. The results show that collapse phenomenon strongly depends on both exerted holding pressure and growth time. The presented model can also give a reasonable prediction of growth and collapse of cells and could give insight to control of cell size in injection foaming process. POLYM. ENG. SCI., 2010. © 2009 Society of Plastics Engineers

Inpp5f Is a Polyphosphoinositide Phosphatase That Regulates Cardiac Hypertrophic Responsiveness

Cardiac hypertrophy occurs in response to a variety of extrinsic and intrinsic stimuli that impose increased biomechanical stress. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has previously been strongly associated with hypertrophic signaling in the heart, and with the control of cell size in multiple contexts. This pathway is tightly regulated by many factors, including a host of kinases and phosphatases that function at multiple steps in the signaling cascade. For example, the PTEN (phosphatase and tensin homolog) tumor suppressor protein is a phosphoinositide 3-phosphatase that, by metabolizing phosphatidylinositol 3,4,5-trisphosphate (PtdIns[3,4,5]P(3), PIP3), acts in direct antagonism to growth factor-stimulated PI3K. Inhibition of PTEN leads to cardiomyocyte hypertrophy. Another polyphoinositide phosphatase, inositol polyphosphate-5-phosphatase F (Inpp5f) has recently been implicated in regulation of cardiac hypertrophy. Like PTEN, this phosphatase can degrade PtdIns(3,4,5)P(3) and thus modulates the PI3K/Akt pathway. To characterize the role of Inpp5f in regulating cardiac hypertrophy. We generated homozygous Inpp5f knockout mice and cardiac specific Inpp5f overexpression transgenic mice. We evaluated their hearts for biochemical, structural and functional changes. Inpp5f knockout mice have augmented hypertrophy and reactivation of the fetal gene program in response to stress when compared to wild-type littermates. Furthermore, cardiac overexpression of Inpp5f in transgenic mice reduces hypertrophic responsiveness. Our results suggest that Inpp5f is a functionally important endogenous modulator of cardiac myocyte size and of the cardiac response to stress.

Cell competition, growth and size control in theDrosophilawing imaginal disc

We report here experiments aimed at understanding the connections between cell competition and growth in the Drosophila wing disc. The principal assay has been to generate discs containing marked cells that proliferate at different rates and to study their interactions and their contribution to the final structure. It is known that single clones of fast-dividing cells within a compartment may occupy the larger part of the compartment without affecting its size. This has suggested the existence of interactions involving cell competition between fast- and slow-dividing cells directed to accommodate the contribution of each cell to the final compartment. Here we show that indeed fast-dividing cells can outcompete slow-dividing ones in their proximity. However, we argue that this elimination is of little consequence because preventing apoptosis, and therefore cell competition, in those compartments does not affect the size of the clones or the size of the compartments. Our experiments indicate that cells within a compartment proliferate autonomously at their own rate. The contribution of each cell to the compartment is exclusively determined by its division rate within the frame of a size control mechanism that stops growth once the compartment has reached the final arresting size. This is supported by a computer simulation of the contribution of individual fast clones growing within a population of slower dividing cells and without interacting with them. The values predicted by the simulation are very close to those obtained experimentally.

Daughter-Specific Transcription Factors Regulate Cell Size Control in Budding Yeast

Author SummaryAsymmetric cell division is a universal mechanism for generating differentiated cells. The progeny of such divisions can often display differential cell cycle regulation. This study addresses how differential regulation of gene expression in the progeny of a single division can alter cell cycle control. In budding yeast, asymmetric cell division yields a bigger ‘mother’ cell and a smaller ‘daughter’ cell. Regulation of gene expression is also asymmetric because two transcription factors, Ace2 and Ash1, are specifically localized to the daughter. Cell size has long been proposed as important for the regulation of the cell cycle in yeast. Our work shows that Ace2 and Ash1 regulate size control in daughter cells: daughters ‘interpret’ their size as smaller, making size control more stringent and delaying cell cycle commitment relative to mother cells of the same size. This asymmetric interpretation of cell size is associated with differential regulation of the G1 cyclin CLN3 by Ace2 and Ash1, at least in part via direct binding of these factors to the CLN3 promoter. CLN3 is the most upstream regulator of Start, the initiation point of the yeast cell cycle, and differential regulation of CLN3 accounts for most or all asymmetric regulation of Start in budding yeast mother and daughter cells.

Selective Deletion of PTEN in Dopamine Neurons Leads to Trophic Effects and Adaptation of Striatal Medium Spiny Projecting Neurons

The widespread distribution of the tumor suppressor PTEN in the nervous system suggests a role in a broad range of brain functions. PTEN negatively regulates the signaling pathways initiated by protein kinase B (Akt) thereby regulating signals for growth, proliferation and cell survival. Pten deletion in the mouse brain has revealed its role in controlling cell size and number. In this study, we used Cre-loxP technology to specifically inactivate Pten in dopamine (DA) neurons (Pten KO mice). The resulting mutant mice showed neuronal hypertrophy, and an increased number of dopaminergic neurons and fibers in the ventral mesencephalon. Interestingly, quantitative microdialysis studies in Pten KO mice revealed no alterations in basal DA extracellular levels or evoked DA release in the dorsal striatum, despite a significant increase in total DA tissue levels. Striatal dopamine receptor D1 (DRD1) and prodynorphin (PDyn) mRNA levels were significantly elevated in KO animals, suggesting an enhancement in neuronal activity associated with the striatonigral projection pathway, while dopamine receptor D2 (DRD2) and preproenkephalin (PPE) mRNA levels remained unchanged. In addition, PTEN inactivation protected DA neurons and significantly enhanced DA-dependent behavioral functions in KO mice after a progressive 6OHDA lesion. These results provide further evidence about the role of PTEN in the brain and suggest that manipulation of the PTEN/Akt signaling pathway during development may alter the basal state of dopaminergic neurotransmission and could provide a therapeutic strategy for the treatment of Parkinson's disease, and other neurodegenerative disorders.

Inhibition of PKHD1 may cause S‐phase entry via mTOR signaling pathway

Autosomal recessive polycystic kidney disease (ARPKD) is an important genetic disorder in pediatric nephrological practice. Mutation of the polycystic kidney and hepatic disease gene 1 (PKHD1) was identified as the cause of ARPKD. Rapamycin is a highly specific inhibitor of mammalian target of rapamycin (mTOR). Rapamycin exerts its biological activity by inhibiting the serine-threonine kinase mTOR, which regulates important cellular processes such as control of cell cycle, cell size, translation initiation and transcription. The ability of rapamycin to cause G1-cell cycle arrest, reduced cell growth and a reduced rate of proliferation has led to efforts to develop rapamycin and related mTOR inhibitors as anti-cystogenesis agents. Therefore, we investigated the relationship between the decreased FPC and the protein levels of mTOR and the inhibitory effects of rapamycin on the expression of mTOR, Hypoxia-inducible factor-1 alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in the human 293T cell line. These observations should provide an important platform for determining FPC function and the pathogenesis of ARPKD, with the targeting of mTOR signaling being exploitable as a novel therapy.

A Rab escort protein integrates the secretion system with TOR signaling and ribosome biogenesis

The coupling of environmental conditions to cell growth and division is integral to cell fitness. In Saccharomyces cerevisiae, the transcription factor Sfp1 couples nutrient status to cell growth rate by controlling the expression of ribosome biogenesis (Ribi) and ribosomal protein (RP) genes. Sfp1 is localized to the nucleus in rich nutrients, but upon nutrient limitation or target of rapamycin (TOR) pathway inhibition by rapamycin, Sfp1 rapidly exits the nucleus, leading to repression of the Ribi/RP regulons. Through systematic cell-based screens we found that many components of the secretory system influence Sfp1 localization. Notably, the essential Rab escort protein Mrs6 exhibited a nutrient-sensitive interaction with Sfp1. Overexpression of Mrs6 prevented nuclear localization of Sfp1 in rich nutrients, whereas loss of Mrs6 resulted in nuclear Sfp1 localization in poor nutrients. These effects were specific to Sfp1 and independent of the protein kinase C (PKC) pathway, suggesting that Mrs6 lies in a distinct branch of TOR and ribosome biogenesis regulation. Rapamycin-resistant alleles of MRS6 were defective in the cytoplasmic retention of Sfp1, the control of cell size, and in the repression of the Ribi/RP regulons. The Sfp1-Mrs6 interaction is a nexus for growth regulation that links the secretory system and TOR-dependent nutrient signaling to ribosome biogenesis.

Overexpression of the downward leaf curling (DLC) gene from melon changes leaf morphology by controlling cell size and shape in Arabidopsis leaves.

A plant-specific gene was cloned from melon fruit. This gene was named downward leaf curling (CmDLC) based on the phenotype of transgenic Arabidopsis plants overexpressing the gene. This expression level of this gene was especially upregulated during melon fruit enlargement. Overexpression of CmDLC in Arabidopsis resulted in dwarfism and narrow, epinastically curled leaves. These phenotypes were found to be caused by a reduction in cell number and cell size on the adaxial and abaxial sides of the epidermis, with a greater reduction on the abaxial side of the leaves. These phenotypic characteristics, combined with the more wavy morphology of epidermal cells in overexpression lines, indicate that CmDLC overexpression affects cell elongation and cell morphology. To investigate intracellular protein localization, a CmDLC-GFP fusion protein was made and expressed in onion epidermal cells. This protein was observed to be preferentially localized close to the cell membrane. Thus, we report here a new plant-specific gene that is localized to the cell membrane and that controls leaf cell number, size and morphology.

Pdk1 activity controls proliferation, survival, and growth of developing pancreatic cells

The formation of adequate masses of endocrine and exocrine pancreatic tissues during embryogenesis is essential to ensure proper nutrition and glucose homeostasis at postnatal stages. We generated mice with pancreas-specific ablation of the 3-phosphoinositide-dependent protein kinase 1 (Pdk1) to investigate how signaling downstream of the phosphatidylinositol-3-OH kinase (PI3K) pathway controls pancreas development. Pdk1-conditional knock-out mice were born with conspicuous pancreas hypoplasia, and within a few weeks, they developed severe hyperglycemia. Our detailed characterization of the mutant embryonic pancreas also revealed distinct temporal, cell type-specific requirements of Pdk1 activity in the control of cell proliferation, cell survival, and cell size during pancreas development. These results thus uncover Pdk1 as a novel, crucial regulator of pancreatic growth during embryogenesis. In addition, we provide evidence that Pdk1 activity is required differently in mature pancreatic cell types, since compensatory proliferation and possible mTORC2 activation occurred in exocrine cells but not in beta cells of the Pdk1-deficient postnatal pancreas.

Cell Size Control: Governed by a Spatial Gradient

The phenomenon of cell size homeostasis, whereby cells coordinate growth and division to maintain a uniform cell size, has been an outstanding issue in cell biology for many decades. Two recent studies in Nature in fission yeast demonstrate that a gradient of the polarity factor Pom1 is a sensor of cell length that determines the onset of Cdc2 activation and mitosis.

A novel Drosophila Girdin-like protein is involved in Akt pathway control of cell size

The Akt signaling pathway is well known to regulate cell proliferation and growth. Girdin, a novel substrate of Akt, plays a crucial role in organization of the actin cytoskeleton and cell motility under the control of Akt. We here identified a novel Girdin-like protein in Drosophila (dGirdin), which has two isoforms, dGirdin PA and dGirdin PB. dGirdin shows high homology with human Girdin in the N-terminal and coiled-coil domains, while diverging at the C-terminal domain. On establishment of transgenic fly lines, featuring knockdown or overexpression of dGirdin in vivo, overexpression in the wing disc cells induced ectopic apoptosis, implying a role in directing apoptosis. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size. Furthermore, this was enhanced by half reduction of the Akt gene dose, suggesting that Akt positively regulates dGirdin. In the wing disc, cells in which dGirdin was knocked down exhibited disruption of actin filaments. From these in vivo analyses, we conclude that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway.

Sin3 is involved in cell size control at Start in Saccharomyces cerevisiae

Saccharomyces cerevisiae cells control their cell size at a point in late G(1) called Start. Here, we describe a negative role for the Sin3/Rpd3 histone deacetylase complex in the regulation of cell size at Start. Initiation of G(1)/S-specific transcription of CLN1, CLN2 and PCL1 in a sin3Delta strain occurs at a reduced cell size compared with a wild-type strain. In addition, inactivation of the transcriptional regulator SIN3 partially suppressed a cln3Delta mutant, causing sin3Deltacln3Delta double mutants to start the cell cycle at wild-type size. Chromatin immunoprecipitation results demonstrate that Sin3 and Rpd3 are recruited to promoters of SBF (Swi4/Swi6)-regulated genes, and reveal that binding of Sin3 to SBF-specific promoters is cell-cycle regulated. We observe that transcriptional repression of SBF-dependent genes in early G(1) coincides with the recruitment of Sin3 to specific promoters, whereas binding of Sin3 is abolished from Swi4/Swi6-regulated promoters when transcription is activated at the G(1) to S phase transition. We conclude that the Sin3/Rpd3 histone deacetylase complex helps to prevent premature activation of the S phase in daughter cells.

Functional interaction of mammalian target of rapamycin complexes in regulating mammalian cell size and cell cycle

Dysregulation of the mammalian target of rapamycin (mTOR) kinase pathway is centrally involved in a wide variety of cancers and human genetic diseases. In mammalian cells, mTOR is part of two different kinase complexes: mTORC1 composed of mTOR, raptor and mLST8, and mTORC2 containing mTOR, rictor, sin1 and mLST8. Whereas, mTORC1 is known to be a pivotal regulator of cell size and cell cycle control, the question whether the recently discovered mTORC2 complex is involved in these processes remains elusive. We report here that the mTORC1-mediated consequences on cell cycle and cell size are separable and do not involve effects on mTORC2 activity. However, we show that mTORC2 itself is a potent regulator of mammalian cell size and cell cycle via a mechanism involving the Akt/TSC2/Rheb cascade. Our data are of relevance for the understanding of the molecular development of the many human diseases caused by deregulation of upstream and downstream effectors of mTOR.

Mechanical Forces of Fission Yeast Growth

Mechanical properties contribute to the control of cell size, morphogenesis, development, and lifestyle of fungal cells. Tip growth can be understood by a viscoplastic model, in which growth is derived by high internal turgor pressure and cell-wall elasticity. To understand how these properties regulate growth in the rod-shaped fission yeast Schizosaccaromyces pombe, we devised femtoliter cylindrical polydimethylsiloxane (PDMS) microchambers with varying elasticity as force sensors for single cells. By buckling cells in these chambers, we determine the elastic surface modulus of the cell wall to be 20.2 +/- 6.1 N.m(-1). By analyzing the growth of the cells as they push against the walls of the chamber, we derive force-velocity relationships and values for internal effective turgor pressure of 0.85 +/- 0.15 MPa and a growth-stalling force of 11 +/- 3 muN. The behavior of cells buckling under the force of their own growth provides an independent test of this model and parameters. Force generation is dependent on turgor pressure and a glycerol synthesis gene, gpd1(+) (glycerol-3-phosphate dehydrogenase), and is independent of actin cables. This study develops a quantitative framework for tip cell growth and characterizes mechanisms of force generation that contribute to fungal invasion into host tissues.