R35GM145248

The dynamics and underlying mechanisms controlling cell size and canonical Wnt signaling - Project Summary/Abstract

Some of the most challenging problems in biology and disease concern dynamical features of the cell. The Wnt pathway is one of the most important developmental and cancer pathways. Control of growth and size is a universal property of all cells, whose dynamics are hard to measure accurately and poorly understood.

The Wnt pathway is made up of conserved scaffolds and enzymes that control the stability of catenin, which regulates important developmental genes. Cell growth control responds to metabolism and differentiation in complex physiological circuits. Components of the Wnt pathway have been long known but how the Wnt signal traverses several kinetic steps before interacting with the catenin is still unclear.

We are trying to understand the Wnt pathway from two approaches. Firstly, through single molecule imaging of fluorescent chimeric proteins knocked into the endogenous loci, thereby preserving the exact level of expression and transcriptional regulation. Secondly, through the development of an in vitro system that preserves the kinetic response of the downstream events of the pathway. From the in vitro system, we can assay purified proteins and assess their activity. We can quickly isolate complexes and study their posttranslational state, and potentially determine the structure of kinetically important forms by cryo-electron microscopy. We have in the past and will in the future combine mathematical modeling with biochemistry to identify key features of this system.

For cell size control, we have used quantitative methods to define the cell's structural and physiological state. We found that mammalian cell size is controlled, not just at G1/S, but throughout the cell cycle by feedback from cell size onto growth rate. How cells know how large they are and regulate their growth is still a mystery. Further understanding will be facilitated by two tools we developed: Computer Enhanced Quantitative Phase Microscopy (CEQPM) and Normalized Raman Imaging (NORI). The former is the most accurate method for measuring cell dry mass for attached cells. The latter can also independently measure protein and lipid mass densities and total mass of cells, even deep within tissues. Furthermore, NORI can measure the rate of protein synthesis and degradation at the single cell level within tissues or in culture in real time.

We will use CEQPM and NORI simultaneously with cultured cells to measure protein synthesis and turnover as a function of cell size and as a function of position in the cell cycle. This will be coupled with pharmacological, growth factor, and nutrient perturbation to identify pathways involved in sensing size and regulating growth. The mechanism of cell size control in differentiated organs under different nutritional states in mouse tissues will also be explored with NORI.

President And Fellows Of Harvard College

THE NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES (NIGMS) SUPPORTS BASIC RESEARCH THAT INCREASES OUR UNDERSTANDING OF BIOLOGICAL PROCESSES AND LAYS THE FOUNDATION FOR ADVANCES IN DISEASE DIAGNOSIS, TREATMENT, AND PREVENTION. NIGMS ALSO SUPPORTS RESEARCH IN SPECIFIC CLINICAL AREAS THAT AFFECT MULTIPLE ORGAN SYSTEMS: ANESTHESIOLOGY AND PERI-OPERATIVE PAIN, CLINICAL PHARMACOLOGY ?COMMON TO MULTIPLE DRUGS AND TREATMENTS, AND INJURY, CRITICAL ILLNESS, SEPSIS, AND WOUND HEALING.? NIGMS-FUNDED SCIENTISTS INVESTIGATE HOW LIVING SYSTEMS WORK AT A RANGE OF LEVELSFROM MOLECULES AND CELLS TO TISSUES AND ORGANSIN RESEARCH ORGANISMS, HUMANS, AND POPULATIONS. ADDITIONALLY, TO ENSURE THE VITALITY AND CONTINUED PRODUCTIVITY OF THE RESEARCH ENTERPRISE, NIGMS PROVIDES LEADERSHIP IN SUPPORTING THE TRAINING OF THE NEXT GENERATION OF SCIENTISTS, ENHANCING THE DIVERSITY OF THE SCIENTIFIC WORKFORCE, AND DEVELOPING RESEARCH CAPACITY THROUGHOUT THE COUNTRY.

93.859 - Biomedical Research and Research Training

National Institute of General Medical Sciences (NIGMS) [HHS - NIH]

HHS Office of the Assistant Secretary for Financial Resources (ASFR) [HHS]

Boston, Massachusetts 021156027 United States

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Maximizing Investigators' Research Award (R35 - Clinical Trial Optional) (PAR-19-367)

Amendment Since initial award the total obligations have increased 318% from $749,190 to $3,129,715.