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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)

HEPATOCYTE POLARIZATION, MITOCHONDRIAL


DYNAMICS AND RESPONSE TO STRESS
Irwin M Arias
Dong Fu, Laszlo Homolya, Yoshi
Wakabayashi, Malte Renz
Jennifer Lippincott-Schwartz
All Lab Members
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

Bile Canaliculi
Scan Electron Microscope

Canaliculi cast
Liver Tissue Section
(ABCB1 staining)

Kamimoto JBC, 1989

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

Bile canalicular network formation


Day1

Day2

Day3

Day4

Occludin
ABCB1

Occludin
ABCB1

Occludin
ABCB1

Occludin
ABCB1

Day5

Day6

***

*** p<0.001
** p<0.01

***
***

***
Occludin
ABCB1

**

Occludin
ABCB1

canaliculi
Hepatocytes
Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Role of LKB1 and AMPK in polarity


LKB1 (Liver Kinase B1) initially identified as a tumor suppressor.
Scaffolding protein

Ser 431 P

LKB1

STRAD

Adaptor protein

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


AMPK alpha unit.
Thr172

AMPK

AMPK

AMPK

AMPK is cellular metabolic sensor and is


activated by AMP.

LKB1-AMPK regulate polarity in various cells


Intestinal epithelial cells
Neurons
Drosophila

Cell. 2004; 116: 457-66


Cell. 2007: 129: 565-77
Nature. 2007; 447: 1017-20

MDCK cells

PNAS, 2007; 104: 819-22

Day 1

Day 1

LKB1

Phos-LKB1

AMPK

Phos-AMPK
-actin

-actin

PNAS 2010
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).

8-Br-cAMP

taurocholate

CPT-cAMP

cAMP

6-Bnz-cAMP
PKI

Epac

LKB1

PKA

canalicular trafficking

AMP

AICAR

ATP

AMPK

AMPK-P
ATP

PP2C

cell
polarization

tight junction formation


cytoskeletal organization

catabolism
lipid synthesis
protein synthesis

AMP

cell growth

Homolya et al (PLOS1, press)

LKB1 and AMPK regulate hepatocyte polarity

Forskolin

Metformin

2-DG

AICAR

Ser 431

MO25

LKB1
STRAD

KD-LKB1 adenovirus

Thr172 P

AMPK

AMPK

AMPK

Myc-DN-AMPK adenovirus

Activation of LKB1 and AMPK accelerates canalicular network formation


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

Forskolin

Occludin
ABCB1

Occludin
ABCB1

2DG

AICAR

Metformin

Occludin
ABCB1

*
*
Occludin
ABCB1

**
*

*
*

Occludin
ABCB1

Fu D. et al. J Cell Sci, 2010

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


formation
Control

GFP

Occludin
ABCB1

GFP
ABCB1

***

LKB1

AMPK

LKB1-KD

Myc-DN-AMPK

LKB1
ABCB1

Myc
ABCB1

***

Canalicular
Formation

Fu D. et al. J Cell Sci, 2010

Immunofluorescence for TOM20 (mito outer-membrane protein)


Day1

Transmission EM
Day 1

Day6

Day2

Day 2

PNAS 2012
Day 6

Dynamic of mitochondrial morphology during canalicular network formation

Fragmented

Tubular

Tubular / vesicular

100

% of cells

80
60

From 3 independent exp.

40
20
Tubular and big vesicles

PNAS 2012

0
Day 0
Fragmented
Tubular
Tubular and big vesicles

Tubular

Day 1

Day 2

Day 3

Fragmented

Day 4

Day 5

Day 6

MITOCHONDRIAL FISSION/FUSION CYCLE

Mfn1, 2
Opa1

DRP1

Mitochondrial fission and fusion protein expressions during canalicular network formation

DRP-1
Phos DRP-1
Actin

Mfn-1
Mfn-2
Actin

Cox IV
Hsp60
Tom20
Actin

Mito Structural proteins

PNAS 2012

PNAS 2012

PNAS 2012

PNAS 2012

PNAS 2012

EPAC/MEK
Bile acid

STRESS (cell isolation,


starvation, drugs, others)

LKB1

NAD-Sirt1-PGCa-FOXO
Thr172
Mito ATP/ FUSION

AMPK

ABCB11: central role


in cholestasis and
polarity

Polarity

* Apical trafficking (rab11aftp1)


* Cytoskeleton arrangement (CLIP170)
* Tight junction formation (PAR 1)

EXCITING DAYS AHEAD.

THANK YOU ALL

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