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UCLA - MCDBIO 165 - Study Guide - Midterm

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UCLA - MCDBIO 165 - Study Guide - Midterm

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background image Aragona M, Panciera T, Manfrin A, Giulitti S, Michielin F, Elvassore N, Dupont S, Piccolo S. 
(2013). A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by 
actin­processing factors. Dev Cell 154(5):1047­59.
A Mechanical Checkpoint Controls Multicellular Growth through YAP/TAZ Regulation 
by Actin­Processing Factors Describes physical/mechanical information signaling through YAP/TAZ to control  proliferation YAP/TAZ regulate cell proliferation in conditions of mechanical stress/tension From discussion: o How do YAP/TAZ respond to their mechanical environment? o Do actin structures transduce signals from the mechanical environment to  YAP/TAZ? o Do contact inhibition and mechanical regulation of proliferation use similar  mechanisms to regulate YAP/TAZ? o How to YAP/TAZ responses to Hippo and other signaling pathways relate to their mechanical roles? Introduction o Cell shape, geometry, and deformations caused by pulling from the  ECM/neighboring cells may cause changes in cytoskeleton/cell tension that signal to  the cell when to proliferate o YAP and TAZ are transcriptional coactivators Go into the nucleus to interact with TEAD transcriptional factors and  increase proliferation o Hippo has been identified as the main input regulating YAP/TAZ o WNT and GPCR signaling also regulate YAP/TAZ o Want to know: Does mechanical regulation of YAP/TAZ regulate growth by  translating the forces the cells experience to the cell? How is this information signaled to YAP/TAZ? Are YAP/TAZ regulators (mechanical, Hippo, WNT, GPCR) interacting  or independent? o Tissue­level control of proliferation of YAP/TAZ depends on F­actin capping and severing proteins CapZ, Cofilin, Gelsolin When there is low mechanical stress, these proteins activity suppress  YAP/TAZ YAP/TAZ­mediated proliferation increases when there is high mechanical stress (stretching or stiff substrate) Results o Mechanical Regulation of Cell Proliferation through YAP/TAZ o Contact Inhibition of Proliferation (CIP) is when cultured cells stop proliferating  when they occupy the entire space allotted to them (when they reach confluence) Regulated by YAP/TAZ When cells are growing, YAP/TAZ are in the nucleus
background image When cells stop growing (as in confluent cultures), YAP/TAZ are in the  cytoplasm In CIP, YAP/TAZ are regulated by phosphorylation In the Hippo pathway, kinases like LATS phosphorylate  YAP/TAZ and keep them out of the nucleus (inactivate them) However, Hippo is not necessary for CIP  o Want to test mechanically regulated CIP Used human mammary epithelial cells (MECs) plated at 3 densities 3,000 cells/cm 2 Sparse No/minimal contact between cells 15,000 cells/cm 2 Confluent All around cell­cell contact 75,000 cells/cm 2 Dense Anti­E­cadherin ( E­CAD) used to show cell boundaries α BrdU is a synthetic nucleotide that shows where newly made DNA is Shows which cells were just made Measures proliferation TOTO3 stains the nucleus Figure 1A shows cells occupy much smaller spaces as density increases
background image Confluent cells show about 30% reduction in proliferation compared to  sparse cells Figure 1B shows increasing cell density greatly reduces proliferation Comparing  BrdU stain with DAPI stain (nuclei stain) shows  α which nuclei were newly made (which cells are new) When YAP/TAZ are knocked down (white bars), there is minimal residual proliferation Shows that not all proliferation is regulated by YAP/TAZ YAP/TAZ activity is causal for S phase entry  YAP/TAZ are antibodies and stains for YAP/TAZ α Black (N) means YAP/TAZ are in the nucleus Grey (N/C) means YAP/TAZ are equally in the nucleus and cytoplasm White © means YAP/TAZ are in the cytoplasm Graph on the right shows how many cells have YAP/TAZ in each  category CIP is reversible Scraping away some of the cells in a monolayer causes cells on the edge to stretch, without losing cell­cell contacts, and cause YAP/TAZ to 
move into the nucleus and then proliferate
background image YAP/TAZ in CIP is also associated with the Hippo pathway Wanted to know if degrees of YAP/TAZ inactivation were  dependent on catenins and LATS Catenins bind cadherin and attach to actin at adherens  junctions Knocking down catenins and LATS rescued YAP/TAZ  transcriptional activity in CONFLUENT cultures Measured by mRNA of YAP/TAZ targets CTGF, CYR61,  and ANKRD1 Knocking these down in DENSE cultures has minimal effects on  YAP/TAZ Since knocking these Hippo genes down in dense cultures had minimal  effects, this likely means YAP/TAZ inhibition is regulated by small cell size If cells are sticking to a small amount of ECM, YAP/TAZ will  inhibit proliferation When cells don't have a lot of substrate to stick to, they have low  mechanical stress Shown by decreased focal adhesions, actin stress fibers,  and cell contractibility  When cells have a lot of substrate to stick to, they  stretch out and form a lot of thick actin stress fibers that 
maintain the tension
MECs plated on small ECM islands mimic the smaller amount of space a  cell in a dense environment would experience Figures 1D and 1E shows that cells plated on small fibronectin (<300um 2 islands stopped proliferating and YAP/TAZ was mostly in the cytoplasm This was independent of cell­cell contact
background image Figures 1F and 1G show that plating cells on a stiff substrate causes cell  proliferation and YAP/TAZ to be in the nucleus Plating on a soft substrate causes cell rounding, YAP/TAZ in the  cytoplasm, and proliferative inhibition Same phenotype from being on soft, dense, or small conditions May relate to YAP/TAZ being controlled by mechanical cues Created a stretching device covered in ECM (fibronectin) Polydimethylsiloxane silicone (PDMS) Seeded cells at high density on PDMS Resulted in YAP/TAZ nuclear exclusion and CIP Stretching caused the adhesive surfaces of the cell to increase 150% Resulted in more actin stress fibers
background image Figure 2B shows 150% increase in adhesive surface of stretched cells DAPI stains the nucleus Figure 2C shows that when the cells are stretched, YAP/TAZ relocate to  the nucleus even in dense cells

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School: University of California - Los Angeles
Department: OTHER
Course: Biology of Cells
Professor: Alvaro Sagasti
Term: Winter 2017
Tags: Microbiology
Name: Paper overviews
Description: These go through the figures of the two papers that are tested on Midterm 2.
Uploaded: 02/20/2017
42 Pages 47 Views 37 Unlocks
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