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Intravital microscopy: a novel �tool to study �membrane traffic in physiological conditions and
during invasion and metastasis �
Roberto Weigert, Ph.D.�Intracellular Membrane Trafficking Unit�Oral and Pharyngeal Cancer Branch�NIDCR-NIH�
Monika Sramkova�
Myo-Pale’ Aye�
Natalie Porat-Shliom�
Sonita Bennett�Laura Parente� Sara Lamb�
Former members�
Intracellular Membrane Trafficking Unit�
PTRU, NIDCR�Thomas H. Bugge�Katiuchia Uzzun Sales�OPCB, NIDCR�Silvio Gutkind�Vyomesh Patel�Kantima Leelahavanichkul�Alfredo Molinolo �VRC, NIDCR�Barton Weick�Rebecca Martinez�NHLBI�Bob Adelstein�Marie Anne Conti�NICHD�Tamas Balla�University of Pittsburgh�Alexander Sorkin�University of South California�Sarah Hamm-Alvarez�
Panomwat Amornphimoltham�
Tim Wigand�
Andrius Masedunskas�
nPan
Kamil Rechache�
Intracellular Membrane Traffic
Endoplasmic Reticulum Golgi Apparatus
Plasma Membrane
Recycling Endosomes
Trans-Golgi Network
Early Endosomes
Late Endosomes Lysosomes
l b
Transport intermediates
Transport intermediate biogenesis Transport intermediate targeting/fusion
Molecular machinery
Aims of the Lab
Golgi Apparatus
Trans-Golgi Network
Recycling Endosomes
Early Endosomes
Endoplasmic Reticulum
s
Early E
Late Endosomes Lysosomes
Plasma Membrane
Cervical lymph-nodes�Primary� tumor�
y p
Golgi Apparatus
Trans-Golgi Network
Regulated exocytosis
Recycling Endosomes
Early Endosomes
Endosomal recycling
Aims of the Lab
Physiopathology of the oral cavity
Plasma Membrane
Animal model�
In vivo�
Explanted organs�
Ex vivo�
3D cell cultures�2D cell cultures�
In vitro�
Physiological relevance�
Manipulation, Reproducibility, Imaging, Accessibility�
Animal model
In vivo
Experimental models to study membrane traffic
1) Biochemical assays
2) Imaging of subcellular organelles
-Electron microscopy
-Light microscopy
Time lapse
Indirect
Static
Intravital Microscopy (IVM)�
Explanted organs
Ex vivo
3D cell cultures2D cell cultures
In vitro
-Light microscopy
Time lapse
Intravital Microscopy�
NIR/IR�
900 nm�
1000 nm�
1100 nm�
1200 nm�
1300 nm�
700 nm�
600 nm�
500 nm�
400 nm�
350 nm�
800 nm�
Single-photon�
E0�
E*�
λ1p�
Two-photon�
λe>λ1p�λ2p�
λ2p=2 λ1p�
Deeper tissue penetration�Limited tissue photo-damage�Limited photo-bleaching�
Technical improvements in�confocal microscopes and optics�
Two-photon microscopy�
Intravital Microscopy�Single-photon�
E0�
E*�
λ1p�
Two-photon�
λe>λ1p�λ2p�
λ2p=2 λ1p�
500 μm�
150 μm�
Two-photon�
60x, NA 1.2�
20x, NA 0.95-1�
1500 μm�
1000 μm�
50 μm�Single-photon�
Deeper tissue penetration�
Homogeneous tissue�
Higher spatial resolution�
Deep tissue�
Long term imaging�Endogenous emission�
50 μm�
140 μm�
700 μm�
Salivary glands �GFP/mTomato mouse �
Tongue Xenograft�GFP-H2B/TX-red dextran/SHG�
Excitation 930 nm�
Brain�TX-red dextran�
Excitation 930 nm�
Tissue�Ti
Resolution�
Intravital Microscopy�
Masedunskas et al., (2008) Traffic, Weigert et al., (2010) Hist. Cell Biol, Amornphimoltham et al., (2010) Adv Drug. Del. Rev�
500
kDa
FIT
C D
extr
an
SG - Parenchyma (740 nm)� SG - Hoechst (820 nm)�
Kidney – Parenchyma (740 nm)�70
kD
a F
ITC
Dex
tran
Liver – Parenchyma (740 nm)�
Tissue� Cellular�Ti C ll l
Resolution�
Intravital Microscopy�
Amornphimoltham et al., (2010) Adv Drug Del. Rev, Bhirde et al., (2009) ACS Nano, Patel et al., (2011) Cancer Research �
Xenograft (Tongue) �
Xenograft (Back)�
OSC
C3-
YFP
Cervical lymph node�
HN
12 (
Hoe
chst
)
Tissue� Cellular�Ti C ll l
Resolution�
Intravital Microscopy�
Sub-cellular�
Amornphimoltham et al., (2010) Adv. Drug Deliv Rev.�
Masedunskas and Weigert (2008), Traffic
500 kDa FITC-Dextran/ 70 kDa TXR-Dextran
1) Stability
2) Spatial resolution
3) Temporal Resolution
4) Quantitative analysis
Early endosomes / Lysosomes
70 kDa 488-Dext / 70 kDa TXR-Dext
An experimental system to image subcellular organelles in live animals
Inverted Upright
Endocytosis of systemically injected fluorescently labeled molecules
………And amenable to pharmacological and genetic manipulations
Masedunskas and Weigert (2008) Traffic - Sramkova et al. (2009), Am. J. Phys - Weigert et al. (2010) Hist. and Cell Bio
1) Delivery of fluorescent molecules
2) Selective delivery of drugs
3) Gene transduction
GFP-Clathrin / TGN38-mcherry
Plasmid DNA
Plasmid DNA/Ad5
Plasmid DNA/PEI
Plasmid DNA/Isoproterenol
Plasmid DNA/Ad5
Cervical lymph-nodes�Primary� tumor�
y p
Golgi Apparatus
Trans-Golgi Network
Regulated exocytosis
Recycling Endosomes
Early Endosomes
Endosomal recycling
Aims of the Lab Plasma Membrane
Regulated exocytosis
Acini
Intercalated ducts
Striated ducts
Salivary glands
TGN
How is the remodeling of the apical membrane, the actin
cytoskeleton and the surface of the granule regulated by a
stimulus originated from the basolateral pole?
Which molecules are activated during secretion…
1) on the surface of the granule?
2) at the apical plasma membrane?
Regulated exocytosis in salivary glands
Stimulus Stimulus
1)
2)
)
GFP-FVB
Mito
trac
ker�
Lyso
trac
ker�
A model to study exocytosis in salivary glands: the GFP mouse
a�
GFP�xy�
Heated pad�
60X water obj.�
Externalized SG�
Microscope stage� Obj. heater�
Coverslip�
e
slip
a Acini�
xz�
Collagen�
Two-photon�
Acinus�
CollCCCC
xy�
15 μm�
Collagen / GFP�xz�
Confocal�
Masedunskas et al., submitted�
Submandibular� Parotid�
Lacrimal� Pancreas�
Sublingual�
Exocrine glands in the GFP mouse�
Masedunskas et al., submitted�
Adrenal�
GFP/Phalloidin�
The architecture of the acini
GFP GFP/Secretory granules�
Secretory granules per acinus� 2300-3100�
Cells per acinus� 9-10�
Diameter of the granules (μm)� 1.5 – 2.0�
Diameter of the canaliculi (μm)� 0.3-0.4�
Masedunskas et al., submitted�
Resting Stimulated – Iso/Carb
Dynamics of the secretory granules during regulated exocytosis in vivo
Apical
pole
al
Masedunskas et al., submitted�
t=0 t=30
Submandibular gland
TGN
Regulated exocytosis in salivary glands
M1/M3 muscarinic receptor
β-adrenergic receptors
Ex-vivo In vivo
β-adrenergic receptors
2) What is the modality of fusion of the
secretory granules in vivo?
1) What is the stimulus required to trigger
exocytosis of the secretory granules in vivo?
Stimulus
allllliiity offfffff ffffffffffffuuuuuuuuuuuussiiiion
Stimulus
mmooooooooooooo
nnnnnnnnn v
sssssssssssss
ccccccrrrrrrrrrrre
ddddddddddddda
vvvvvvvvvvvviiiiiiiiiiiiv
tttttttttiiiiiiiiiiiiiim
eeeeeeeeeeetttttttttt
gggggggggggggeeeeeiiigggggggggggg
What is t
tttttoooooorrrrrryyyyyy ggggggrrrrrraanu
oc f th
creto
What is
tttooory grr
occccccccccccccccccyyyyyyyyyyyyyyyyyttttttttttosis oooooooooooooooooooofffffffffffffffffff xo
Single fusion event Kiss-Run event Compound exocytosis
3) How is fusion regulated?
Signaling through β-adrenergic and not muscarinic receptors stimulate the exocytosis of the
secretory granules in the salivary glands in vivo
Masedunskas et al., submitted�
0:00 0:10 0:21 0:29
0:36 0:40 0:45 0:48
0:56 1:08 1:28 1:48
0:00
Fusion pore Apical pole
Secretory granules exocytosis in vivo occurs through single fusion events
Masedunskas et al., submitted�
Sec Granule Plasma membrane
10 kDa Texas-Red Dextran�
Masedunskas et al., submitted�
Secretory granules exocytosis in vivo occurs through single fusion events
Tomato- MGCCFSKT
PM bilayer
Extr
acel
lula
r
Cyto
pla
sm
Masedunskas et al., submitted�
KTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT KTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
Collapse Membrane diffusion
t=5-10 sec
Sec Granule Plasma membrane
t=5-10 sect=0 t=40-90 sec
Secretory granules exocytosis in vivo occurs through single fusion events
Φ=1.5 μm� Φ=0.3 μm�
Tom
ato�
Phal
loid
in�
GFP-Lifeact (F-actin)�
Riedl et al., (2008) Nat. Methods�Courtesy of Tamas Balla (NICHD)�
Actin is recruited onto the secretory granules after fusion with the APM �
Masedunskas et al., submitted�
Ctr
l�Is
o�
GFP
-Lif
eact
�GFP-Lifeact�
Iso�
GFP-Lifeact mouse�
Time (seconds)�
Actin is recruited onto the secretory granules after fusion with the APM �
0:00� 0:01� 0:03� 0:05�
RFP
-Far
n�G
FP-L
ifea
ct�
Sec Granule� Canaliculus� Actin�
Actin nucleation and polymerization�
Collapse�
))))))))))))))))))))))seconnndddddddddddddddddddddddddddddddddsssssssssssssssssssssssssssssssssssss))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee TTTTTTTTTTTTTTTTTTTTTTTiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiimmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
RFP-Lifeact�GFP-Farnesyl�C
trl�
Iso�
Masedunskas et al., submitted�
Cytochalasin D
Actin is required to complete the collapse of fused the secretory granules
Contr
ol
Masedunskas et al., submitted�
0:00� 0:05� 0:27� 0:35� 0:37�
0:39� 1:05�0:45� 1:35� 3:05�
Isopro
tere
nol
Cytochalasin D
β-adrenergic�stimulation�
gic
Hydrostatic pressure�
Masedunskas et al., submitted�
GFP Phalloidin 0:00� 0:01� 0:02� 0:03� 0:14�
0:22� 0:31� 0:32� 0:36� 0:42�
Actin is required to complete the collapse of fused the secretory granules
Myosin motor
Myosin IIa and IIb are recruited onto the secretory granules
Masedunskas et al., submitted�
Myo IIb/Phalloidin�
Myo IIa/Phalloidin�
Isopro
tere
nol
Myo IIa�
Myo IIb�
Contr
ol
Bhat and Thorn, (2009) MBC�
Pancreatic acini�
Actin is required to complete the collapse of fused the secretory granules
Isoproterenol Control
GF
P-m
yosi
n I
Ib
Andrius Masedunskas, Bob Adelstein, Marie Anne Conti�
Myosin IIa and IIb are recruited onto the secretory granules T
ime
Isop Ctrl Kymograph
GF
P-m
yosi
n I
Ia
GF
P-m
yosi
n I
Ib
(-)
bleb
bist
atin
�(+
) bl
ebbi
stat
in�
m-T
omat
o�Iso�Control�
The impairment of the motor activity of myosin II affects the collapse of the SCGs
0:00� 0:03�0:01� 0:12�
0:22� 0:34� 0:43� 1:05�
0:00� 0:05�0:01� 0:10�
0:18� 0:35� 0:36� 0:53�
*�
Masedunskas et al., submitted�
Hydrostatic� pressure�
β-adrenergic�stimulation�
Actin�
Actin� β-adrenergic�stimulation� Myosin IIa/IIb�
Canaliculus�
SCGs�
Model
Φ=0.3 μm�
Φ=1.5 μm�
Higher curvature�
Lower curvature�
Φ=0.3 μm
Φ=1.5 μm
Higher curvature
Lower curvature
Cervical lymph-nodes�Primary� tumor�
iiiiiiiicccccccaaaaaaaallllllllll llllllllllllllllllllllllyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyymmmmmmmmmmmmmmmppppppppppppppppppppppppppppphhhhhhhhhhhhhhhhh----nnnnnnooooooooddddddddddddeeeeeeeeeeeesssssCCCCCCCCCCCCCCCCCCCeeeerrrrvvvvPrimaryyyyy tumor
yy ppppppppppppppppppp
• Sixth most common cancer in the developed world (500,000 new cases; 250,000 deaths/year)
• 37,000 new cases of head and neck cancer/year (8,000 deaths/year) in U.S. (Cancer satistics, 2010)
• The incidence of oral cancer varies greatly worldwide • 90-95% are squamous cell carcinoma • 30-40% are originated from dorsal and lateral tongue • Survival rate less than 50%
Head and Neck Cancer�
Role of membrane trafficking in metastasis
Cell motility Endosomal recycling
Early endosomes
Recycling endosomes
Invasion and metastasis
Directing molecules to specific locations of the PM
Adhesion
Signaling
Matrix degradation
Moving membranes in the direction of migration
1) Which recycling pathway controls the invasion process ?
2) How is invasion regulated by endosomal recycling ?
What is the role of membrane trafficking in invasion and metastasis?�
Cervical lymph-nodes�Primary� tumor�
y p
E
What is the role of membrane trafficking in invasion and metastasis?�
Early endosomes
Recycling endosomes
E
Endosomal recycling Rab GTPase
Rab4
Rab11a,b
Rab25
Rab22
Rab8
Downregulated in breast cancer �(Cheng KW et al., 2006)�
Overexpressed in breast and ovarian cancer�(Cheng JM et al., 2004)�
Interacts with integrin α5β1�(Caswell PT et al., 2007)�
Downregulated in colon cancer�(Nam KT et al., 2010)�
Close homologue of Rab11a and Rab11b�
Implicated in recycling in epithelial polarized cells �
RRRRRRRRRabbbbbbbbb222222222555555555 b
The small GTPase Rab25 is down regulated in human HNSCC tissues�
Normal� Tumor�
0�
2�
4�
6�
8�
10�
Stai
ning
sco
re�
T test : p<0.0001�
N=23
N=175
Lymph nodes metastasis�
0�
2�
4�
6�
8�
10�
N=23�
ANOVA: p<0.0001�
***�***�
***�***�
***�
N=70�
N=29�
N=6�
N=49� N=21�
Normal� Tumor� Normal� T1� T2� T3� T4� Tx�
Amornphimoltham et al., man in prep
Nude or Scid mice�
Xenograft in the mouse tongue
Experimental model�
Patel et al., (2011) Cancer Research, Amornphimoltham et al., man in prep
Cervical lymph-nodes�Primary� tumor�
ph-nodesCCCCCCCPrimaryy tumor
vicalalalalllalal llll llllymymymymymymymymphphphphphphphphCeCCCCCCC rv
Primary� tumor� Cervical lymph-nodes�
EGFR�
Rab25�
Cal
27�
UM
SCC
2�
UM
SCC
17B
�
OSC
C3�
OR
L48
�
OR
L15
0�
HeL
a C
CL
2�
HeL
a #1
�
HN
12�
OSC
C3
O
OSCC3� HeLa� #3�
H2B/ Endog fluo / Stromal cells�
Nude or Scid mice�
Cervical lymph-nodes�
Primary� tumor�
Xenograft in the mouse tongue
Experimental model�
Excitation 740/930 nm�
Excitation 930 nm�
Excitation 930 nm�
H2B/ Collagen/ Lymphatic�
Patel et al., (2011) Cancer Research, Amornphimoltham et al., man in prep
H2B/ Dextran�
Pri
mar
y tu
mor
�C
ervi
cal l
ymph
nod
e�Hela#3�venus�
Hela#3�venus-RAB25�
Per
cent
age
of m
ice
wit
h tu
mor
bur
den�
0�
20�
40�
60�
80�
100�
primary tumor� LN metastasis�
Rab25 re-expression blocks invasion and metastasis in vivo�
Hela#3-Venus Rab25/ Hela#3-mCherry/ Lyve1�
Amornphimoltham et al., (Man. in prep.)
Hela#3-Venus�Hela#3-Venus Rab25�
Hela#3-Venus � Hela#3-Venus Rab25/ Hela#3-mCherry�
Rab25 re-expression blocks invasion and metastasis in vivo�
Day 23 24 27 25 26
Amornphimoltham et al., (Man. in prep.)
Rab25 plays an important role in preventing invasion and metastasis�
What is the mechanism?�1) Pro-apoptotic�2) Prevent angiogenesis�3) Regulate cell cycle�4) Cell motility�5) Adhesion�
1111111111111))))))))))) PPPPPPPPPPPPrrrrrrrrrrrroooooooooooo----aaaaaaaaaaaaappppppppppppoooooooooooopppppppppppttttttttttttooooooooooottttttttttiiiiiiiiiicccccccccccc222222222222))))))))))) PPPPPPPPPPrrrrrrrrreeeeeeeeevvvvvvvvveeeeeeeeennnnnnnnnttttttttt aaaaaaaaannnnnnnnngggggggggiiiiiiiiioooooooooogggggggggggeeeeeeeeeeennnnnnnnnnneeeeeeeeeeesssssssssssiiiiiiiiiiisssssssssss3333333333333))))))))))))) RRRRRRRRRRRRReeeeeeeeeeegggggggggggguuuuuuuuuullllllllllllaaaaaaaaaattttttttttteeeeeeeeeee ccccccccccceeeeeeeeeeeelllllllllllllllllllll ccccccccccyyyyyyyyyyyccccccccccccclllllllllleeeeeeeeeeeee
Early endosomes
Recycling endosomes
E
Rab4
Rab11a,b
Rab25
Rab22
Rab8
RRRRRRRRRabbbbbbbbb222222222555555555 b
Venus Rab25�
Integrin trafficking?
Binding to β1-Integrin�
Rab25�
Rab11�
Rab25>11L�
Rab11>25L�
170 213
yes�no�no�yes�
No effect of Rab25 on integrin localization or trafficking�
The C-terminus of Rab25 is necessary and sufficient to block cell invasion in vitro�
Amornphimoltham et al., (Man. in prep.)
Perc
enta
ge o
f m
ice
with
tum
or b
urde
n�
0�
20�
40�
60�
80�
100�
primary tumor� LN metastasis�
ns�**�
***�ns�
RAB25�RAB25>11L�RAB11>25L�
Venus�
Caswell PT et al., 2007�
Rab25 plays an important role in preventing invasion and metastasis�
What is the mechanism?�1) Pro-apoptotic�2) Prevent angiogenesis�3) Regulate cell cycle�4) Cell motility�5) Adhesion�
1111111111111))))))))))) PPPPPPPPPPPPrrrrrrrrrrrroooooooooooo----aaaaaaaaaaaaappppppppppppoooooooooooopppppppppppttttttttttttooooooooooottttttttttiiiiiiiiiicccccccccccc222222222222))))))))))) PPPPPPPPPPrrrrrrrrreeeeeeeeevvvvvvvvveeeeeeeeennnnnnnnnttttttttt aaaaaaaaannnnnnnnngggggggggiiiiiiiiioooooooooogggggggggggeeeeeeeeeeennnnnnnnnnneeeeeeeeeeesssssssssssiiiiiiiiiiisssssssssss3333333333333))))))))))))) RRRRRRRRRRRRReeeeeeeeeeegggggggggggguuuuuuuuuullllllllllllaaaaaaaaaattttttttttteeeeeeeeeee ccccccccccceeeeeeeeeeeelllllllllllllllllllll ccccccccccyyyyyyyyyyyccccccccccccclllllllllleeeeeeeeeeeee
Early endosomes
Recycling endosomes
E
Rab4
Rab11a,b
Rab25
Rab22
Rab8
RRRRRRRRRabbbbbbbbb222222222555555555 b
Cytoskeleton
Amornphimoltham et al., (Man. in prep.)
Venus Rab25/ Phalloidin�Phalloidin�
Hel
a#3-
Ven
us
Hel
a#3-
Ven
us R
ab25
Rab25 re-expression reduces actin-rich structures at the PM �
Cells migrating in 3D�
Hel
a#3-
GFP
-lif
eact
Rab25 re-expression reduces actin-rich structures at the PM �
Recycling endosomes�
Early endosomes�mmesmes
Actin
Recycling endosomes�
Early endosomes�mmesmes Rab25
Negative Regulator
Rab25
MAY 18-19, 2011�