Irwin M. Arias, MD Triple Emeritus: Professor of Medicine (Albert Einstein College of Medicine); Professor of Physiology and Medicine (Tufts University School of Medicine and Senior Scientist (National Institute of Health)

Kim Brouwer, UNC. Neal Ruderman, BU., Sangeeta Bhatia, MIT, Lewis Cantley, Harvard Johann Auwerx, Lausanne,Reuben Shaw, Salk,Lee Hagey, UCSD, Silvio Gutkind, NIDCR, Mary Ann Conti,, NHLBI, Marcello Amara, NHLBI

Unit of Cellular Polarity, Cell Biology and Metabolism Program, NICHD, National Institutes of Health, Bethesda, MD 20817 ariasi@mail.nih.gov

HEPATOCYTE POLARIZATION, MITOCHONDRIAL DYNAMICS AND RESPONSE TO STRESS

Dong Fu, Laszlo Homolya, Yoshi Wakabayashi, Malte Renz Jennifer Lippincott-Schwartz All Lab Members

Irwin M Arias

Loss of polarity results in bile secretory failure (cholestasis) and cell death.

Scan Electron Microscope

Liver Tissue Section (ABCB1 staining)

Canaliculi cast

Bile Canaliculi

Kamimoto JBC, 1989

Day2 Day1 Day3

Day5 Day6

Occludin ABCB1

Occludin ABCB1

Occludin ABCB1

Occludin ABCB1

Occludin ABCB1

** ***

***

*** *** *** p<0.001

** p<0.01

Day4

Occludin ABCB1

Bile canalicular network formation

Day 2

Hepatocytes

canaliculi

Day 3 Day 4 Day 5 Day 6 Day 1

Role of LKB1 and AMPK in polarity LKB1 (Liver Kinase B1) initially identified as a tumor suppressor.

LKB1 activates AMP-activated protein kinase (AMPK) by phosphorylating threonine 172 on AMPK alpha unit.

AMPKα

AMPKβ AMPKγ

P Thr172

AMPK is cellular metabolic sensor and is activated by AMP.

Intestinal epithelial cells Cell. 2004; 116: 457-66 Neurons Cell. 2007: 129: 565-77 Drosophila Nature. 2007; 447: 1017-20 MDCK cells PNAS, 2007; 104: 819-22

LKB1-AMPK regulate polarity in various cells

STRAD LKB1

Scaffolding protein

Adaptor protein P Ser 431

B

C

Phos-AMPK AMPK Phos-LKB1 LKB1

Day 1 2 3 4 5 6

β-actin β-actin

A Day 1 2 3 4 5 6

Fig2: Dynamic of protein levels of total and phosphorylated LKB1 and AMPK. A: Western blot for LKB1, AMPK, phos-LKB1

and phos-AMPK. B, C: Densitometery measurements (n= 4).

PNAS 2010

canalicular trafficking

cell polarization

AMPK

AMP ATP

LKB1

AMP

ATP PP2C

lipid synthesis

protein synthesis

catabolism

cAMP

Epac

taurocholate

PKA

AMPK-P

AICAR

CPT-cAMP 6-Bnz-cAMP

PKI

cell growth

8-Br-cAMP

tight junction formation

cytoskeletal organization

Homolya et al (PLOS1, press)

AMPKα

AMPKβ AMPKγ

P Thr172 MO25

STRAD

LKB1

P Ser 431

Forskolin Metformin 2-DG AICAR

KD-LKB1 adenovirus Myc-DN-AMPK adenovirus

LKB1 and AMPK regulate hepatocyte polarity

**

** * **

*

AICAR

Metformin control

Occludin ABCB1

Occludin ABCB1

Occludin ABCB1

Occludin ABCB1

Forskolin

Occludin ABCB1

2DG

Activation of LKB1 and AMPK accelerates canalicular network formation

Day 2 cells are treated with various activators for 24h.

Fu D. et al. J Cell Sci, 2010

Infection with KD-LKB1 or DN-AMPK viruses inhibit canalicular network formation

Control

Occludin ABCB1

GFP

GFP ABCB1

LKB1-KD

LKB1 ABCB1

Myc-DN-AMPK

Myc ABCB1

*** ***

Fu D. et al. J Cell Sci, 2010 LKB1 AMPK

Canalicular Formation

Immunofluorescence for TOM20 (mito outer-membrane protein) Day2 Day1 Day6

Transmission EM

Day 2 Day 6 Day 1

PNAS 2012

Dynamic of mitochondrial morphology during canalicular network formation

Day 0 Day 1 Day 2 Day 3Day 4

Day 5Day 6

Fragmented

Tubular

Tubular and big vesicles0

20

40

60

80

100

FragmentedTubularTubular and big vesicles

Fragmented Tubular Tubular / vesicular

From 3 independent exp.

% o

f cel

ls

PNAS 2012

MITOCHONDRIAL FISSION/FUSION CYCLE

DRP1

Mfn1, 2 Opa1

Mitochondrial fission and fusion protein expressions during canalicular network formation

DRP-1

Actin

Phos DRP-1

Mfn-1

Mfn-2

Actin

Cox IV

Hsp60

Tom20

Actin

Mito Structural proteins

PNAS 2012

PNAS 2012

PNAS 2012

PNAS 2012

PNAS 2012

AMPK

Polarity

LKB1

Mito ATP/ FUSION

Bile acid

* Apical trafficking (rab11aftp1) * Cytoskeleton arrangement (CLIP170) * Tight junction formation (PAR 1)

EPAC/MEK

Thr172 NAD-Sirt1-PGCa-FOXO

STRESS (cell isolation, starvation, drugs, others)

ABCB11: central role in cholestasis and polarity

EXCITING DAYS AHEAD………….

THANK YOU ALL