presentación de powerpoint - seqc · anabolism catabolism. ubiquitin proteasome system main...

Autofagia: el sistema de reciclaje que

necesitan todas nuestras células

CIB-CSIC, Madrid, Spain

[email protected]

CSICCONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS

Caloric restriction

Caffeine

Green Tea

Rapamycin

Resveratrol

Metformin

Spermidine

Lithium Exercise

Hypoxia

Torin-1

Trehalose

What have all these treatments have in common?

Anti-aging

Caloric restriction

Caffeine

Green Tea

Rapamycin

Resveratrol

Metformin

Spermidine

Lithium Exercise

Hypoxia

Torin-1

Trehalose


Autophagy

• ¿Qué es? Tipos / Métodos estudio

• Funciones: implicaciones en fisiología

• Enfermedad y Envejecimiento

Autofagia

Anabolism Catabolism

Ubiquitin proteasome system

Main catabolic pathways in cells

Autophagy-lysosomal system

Lysosome

Acid

hydrolases

Autofagia

Autofagia

Auto

Fagia= comer

Proceso de digestión intracelular y

reciclaje de los componentes celulares

Discovery of autophagy

Christian de Duve 1974

Lysosomes

Autophagosomes

Autophagy 1963

Atg genes 1992

Yoshinori Ohsumi 2016

Catabolic mechanism

Regulated by

hormones

15’

1h

2h

3h

Proteinase

Deficient yeast

Nutrient deprivation

Functional Groups of Atg Proteins(Y. Ohsumi, M Thumm, DJ Klionsky)

1. Protein Kinase Complex

Atg1, Atg13 , Atg17 , Atg11

2. PI3 Kinase Complex

Atg6 (Beclin 1), Atg14 , (Vps15 , Vps34)

3. Recycling pathway, others

Atg2 , Atg9 , Atg18

4. Atg12 Conjugation System

Atg12 , Atg7 , Atg10 , Atg5 , Atg16

5. Atg8 Lipidation System (PE)

Atg8 (LC3), Atg4, Atg7 , Atg3

Initiation

Nucleation

Expansion

Sealing

Atg genes

Diseases

Neurodegeneration

Muscle disorders

Aging

Infectious diseases

…

Autophagy: the molecular era

Boya P, et al. Nature Cell Biology (2013) 15:713-20

Types of Autophagy

Macroautophagy

Molecular regulators: Atg proteins

Selective autophagy

Methods to detect autophagy

+E64d

Tubulin

LC3 I

LC3 II

0.5 2 4 60EBSS (h)

Autophagic flux

2. Increase in LC3 puncta

CO EBSS

GFP-LC3 transfection IF anti-LC3 antibody

CO Induction

LC3-GFP

Eskelinen 2005

New tools for quantitative Autophagy

Image analysis SISTEMA EXPERTO DE ANÁLISIS CUANTITATIVO DE IMÁGENES PARA EVALUACIÓN AUTOMATIZADA DE ENSAYOS CELULARES DE AUTOFAGIA Y APOPTOSIS

WimScratch

for Wound Healing Assay

WimTube

for Tube Formation Assay

WimTaxis

Automated Cell Tracking

WimSprout

for Sprouting Assay

WimCounting

for cell and nuclei counting nuclei

WimAutophagy

Autophagy in cells and tissues

WimApoptosis

Apoptosis in cells and tissues

WimAutophagy


5.LC3 puncta quantification

Tamaño (px)

Frecuencia

Tamaño medio autofagosoma: 8 px

Tamaño mín autofagosoma: 3 px

Tamaño máx. autofagosoma: 11 px

Tamaño cluster: >12 px

AP

Cluster

WimAutophagy


Cells transfected with GFP-LC3Cells transfected with the Tandem GFP-RFP-LC3Immnunodetection of LC3 with antibodies

Epiflourescence/confocal microscopy

WimAutophagy


HeLa si RNA scramble-GFPsi RNA scramble Atg7-GFP

Ct N/L

20

60

Num

eb

r o

f a

uto

phago

som

e/ ce

ll

Ct N/L

100

*Scramble

Atg7 KD

*

GFP

Funciones de la autofagia

Physiological functions of autophagy

Basal autophagyInduced autophagy

Degradation of protein aggregates



Elimination of damaged organelles

Elimination superfluous organelles



Cell viability during starvation


Metabolic adaptation






Degradation of intracelullar pathogensAntigen presentation











Protection against DNAdamage


Cell death



Diseases associated to altered autophagy





Protection against DNAdamage


Cell death


Cancer

Neurodegeneration

Infectious diseases

Chronic inflammation

Heart diseases

Cancer

Cancer

Aging

Diabetes, obesity

Autophagy

Respuesta adaptativa al estrés

Mecanismo citoprotector P Boya et al., Mol Cell Biol 2005

González-Polo et al., J Cell Sci 2005

HCQ

Monensin

Bafilomicin A1

Lamp 1,2 -/-

siRNA Lamp1,2

3-MA

siRNA Atgs

Degradación intracelular: autofagia selectiva

Caloric restriction

Caffeine

Green Tea

Rapamycin

Resveratrol

Metformin

Spermidine

Lithium Exercise

Hypoxia

Torin-1

Trehalose


Anti-aging

Lausanne octobre 2008

Caloric restriction (rhesus monkeys):

Colman et al. 2009 Science 325, 201

Rapalogues and TOR inhibitors (mice, drosophila):

Harrison et al. 2009 Nature 460, 392

Lee et al. 2010 Science 327, 1223

Autophagy and anti-ageing

Autophagy and anti -Ageing

Aging = decreased autophagic pathways

- Fusion problems (reduced macroautophagy and CMA)

- Decreased mRNA expression of Atg genes

- Accumulation of pigments--- Lipofuscin

- Inadequate turnover of damaged mitochondria---- ROS

- Energy compromise

Beneficial effects of autophagy on Aging

Autophagy

Damaged

mitochondria

Aggregated

proteinsER-stress

ROSproduction Lipolisis Increase

ATP production

Decreased Ageing

Autophagy

Damaged

mitochondria

Aggregated

proteinsER-stress

ROSproduction Lipolisis Increase

ATP production

Decreased Ageing

Take home message!

Alumni

Lorena Esteban-Martinez

Lucia Garcia Ledo

Esther Seco Martin

Fengjuan Wang

Marta Mauro

Rita Marques

Kike Iglesias

Marián Mellén

Patricia Vázquez

Sergio Hernández

Marta Fierro

Current members

Elena Sierra-Fliardi

Ana Serrano Puebla

Beatriz Villarejo Zori

Raquel Gomez Sintes

Ignacio Ramirez Pardo

Natalia Rodriguez-Muela

presentación de powerpoint - seqc · anabolism catabolism. ubiquitin proteasome system main...

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