Cellular hypertrophy Control of protein accretion –Endogenous Nutrient status Functional adequacy History –Exogenous Nutrient availability Growth factors Space Protein synthesis (translation) Protein degradation (autophagy)

Immediate controls on protein synthesis Substrate availability –m-/t-RNA –Amino acids –Phosphatidylethanolamine (ATG8/LC3) Catalyst availability & activity –rRNA & S6 phosphorylation –eIF2, 4 –eEF1, 2

Major regulatory pathways Positive –Akt: mTORc, inhibit FOXO –mTORc Growth Factors, nutrients, hypoxia S6k, 4E-BP1, inhibit ULK-1 Negative –AMPK AMP/ATP, calcium ULK-1, TSC1/2, eEF2K –FOXO (transcription factors) Ubiquitin ligase, glutamine synthase (autophagy)

Signal integration through mTOR TSC2 TSC1 RAPTOR Ragulator RAG Lysosome Rheb RAPTOR GTP GDP RAG GDP PRAS40 LST8 PRAS40 LST8 mTOR Amino Acids Insulin (GF) Hypoxia ATP/AMP ERK Akt AMPK GSK3 REDD Adhesion Akt

Signal integration through AMPK ATP/AMP GSK3 mTOR eEF2K TFE Autophagy Genes Protein Synthesis eIF2b p70 etc eEF2 eIF2a Ca2+ CaMKK LKB1 STRAD MOL25a ATM DNA Damage Sympathetic NS PKA Glucose uptake Lipolysis Mitochondria AMPK  AMPK  folliculin AMPKa FNIP

Stimuli for size change Developmental –Strongly dependent on growth factors –Feedforward Functional –Overload –Endogenous factors –Feedback Integrative –Autocrine/paracrine factors

Linking growth stimuli to growth Insulin/IGF-1 (growth factors, generally) –IGF1  IGF1R  IRS1  Grb2  SOS  Ras  Raf  MEK  ERK  MNK--|4EBP1  PI3K PI3K –IGF1/FAK  PI3K  PIP3  PDK1  akt --|TSC--|Rheb  mTOR  RSK  S6  translational apparatus --|4EBP--|eIF4E  translational activity --|eEF2K--|eEF2  translational activity --|GSK3b--|eIF2  translational activity

Physical niche is important Adhesion/turnover –Integrin  FAK  Src/paxillin  PI3K  Akt  mTOR  c-Myc –Transcriptional targets include eIFs, eEFs, tRNA synthetases, Pol III, nucleolar proteins, 40S & 60S proteins –  CTD phosphorylation, mRNA cap methylation  ERK1/2 Huang & Ingber 1999 Cells grown on different attachment areas. Yellow  synthesizing DNA Blue  not synthesizing DNA

Negative controls on growth AMPK –AMP  AMPK  TSC--|mTOR --|IRS1  GSK3b--|eIF2 FOXO, esp FOXO3 –Oxidative/heat stress/Ppar-g  FOXO  p27Kip--|cell cycle  MuRF, atrogin  ubiquitinylation  myostatin (FOXO1) TGF-b –TGFR  SMAD  apoptosis, cell cycle arrest…

Paracrine signaling Positive –PGE2, PGF2a –GH/IGF-1 axis Negative –TGF-b family

Functional influences Muscle –Mechanical overload  hypertrophy –Cell damage (creatine kinase) –Metabolite coupling (PPAR  ) Liver –Toxin overload  hypertrophy+hyperplasia –Cell damage (alanine aminotransferase) –Metabolite demand (PPAR  ) Epithelium –Competitive hypertrophy –Contact inhibition

Cell size transduction Mass action –Cube-square metabolite accumulation –Finite RNA/protein synthesis rates –Nucleus/cytoplasm volume (sc Cln3) –Diffusion (polar expression of Pom1) Contact –Cadherin--|b-catenin  cell cycle –Hippo/MST1/2--|Yki/Yap  cell growth

Integrative control Mechanical demand –FAK, Ca2+, other force sensors Energetic demand –AMPK, GSK, Ragulator Net growth of cell depends on balance –Overloaded, high energy cell grows –Overloaded, starved cell atrophies –Infrequent overload is sufficient to maintain or gain functional capacity mTOR is a signal integrator