Transcript Cynthia Santoso`s HHMI Project

Calcium regulation on a-actinin
by
Cynthia Santoso
Dr. Jeffrey Greenwood Lab
Biochemistry and Biophysics Department
Cancer

Cancer is a group of diseases
characterized by uncontrolled growth and
spread of abnormal cells.

If the spread is not controlled, it can result
in death.
Cancer

Cancer is caused by both:
external factors (tobacco, chemicals,
radiation, infectious organisms)
 Internal factors (inherited mutations,
hormones, immune conditions, and mutations
that occur from metabolism)

Who is at risk of developing
cancer?

ANYONE.

Since occurrence of cancer increases as
individuals age, most cases affect adults
beginning in middle ages.
Cancer

Types of cancer:

Benign tumor
- Remains confined to its original location, neither
invading surrounding normal tissue nor spreading
to distant body sites.

Malignant tumor
- Capable of both invading surrounding normal
tissue and spreading throughout the body via the
circulatory or lymphatic system.
Cancer

Only malignant tumors are properly
referred as cancers, and it is the ability to
invade and metastasize that makes cancer
so dangerous.
* metastasis refers to the spread of cancer
cells from their site of origin to other sites
in the body.
Metastasis
• The very important process by
which cancer spreads from one
part of the body to another.
• How do these cancer cells
move about?
Metastasis




Moving cells, whether healthy or
cancerous, grab the extracellular
matrix with tiny feet, called focal
adhesion complexes.
They move their way up along the
extracellular matrix.
Upon reaching a vein or artery,
they have a long trip throughout
the body.
At the end of the journey, they exit
the blood vessel and found a new
location to further grow.
Cell Adherence
Attached
Spread
Cell with stress fibers
Cell
Cell
and focal adhesions
De-adhesion
Weak
Adherence
Intermediate
Adherence
Strong
Adherence

Cell adhesion is an important mechanism by which cells
interact with the extracellular environment.

The adhesive state of a cell has significant influence on
growth and survival, migration, and signal transduction.
Focal Adhesion Structure
a-actinin
a-actinin
a-actinin
a-actinin
a-actinin
a-actinin
a-actinin
Vinculin
Talin
b a
a-actinin
a b
FAK
Talin
Paxillin Talin
b a
a b
Talin
b a
Extracellular Matrix
a-actinin
a b
Syndecan-4
a-a
a-a
PDGF treated fibroblasts
a-a
a-a
P
Strong Adherence cell with stress fibers
and focal adhesions
T V
T
V
b a
PDGF
a-a
a-a
a b
a b
a b
PI 3-kinase
a-a
V
V
Intermediate
Adherence
= PtdIns (3,4,5)-P3
T
a b
a-a
P
a b
a b
a b
T
Hallmark of cell adhesion and
motility studies

It enables us to modulate or adjust or vary
these signals in order to control both
desirable and undesirable cell growth and
motility.
Alpha-actinin structure
1
CH1
141
256
CH2
EF hands
364
479
600
1
2
3
4
3
2
713
887
4
1
Spectrin repeats
CH2 CH1
ABD

a-actinin is an anti-parallel homodimer.

Each monomer is composed of three domains:



The actin-binding domain
The spectrin repeats
The C-terminal EF hands domain
Fact
•
•
It has been known that calcium regulates
a-actinin bundling activity.
“Calcium oscillation trigger focal adhesion
disassembly…” by Giannone et al.
Question
1. Do the EF Hands domains bind
specifically to the Actin-binding domains?
2. If so, is the binding regulated by Calcium
ion?
The Young et al Hypothesis

The closed or inactive state of the
molecule exists when the EF 3/4 region of
the CaM-like region of the CaM-like
domain interacts with a region between
the ABD and R1 of the opposite subunit.
Ca2+
Ca2+
The Tang et al Hypothesis

In the presence of Ca2+, the CAL domain (EF
Hands) of a -actinin could undergo a
conformational change that enables it to wrap
around… the connecting helix between the
two CH domains in the ABD.
Ca2+
Ca2+
Method
Protein-protein overlay assay.
 We did overlay with the full length of aactinin.

Protein-Protein Overlay Assay
Enhanced
Chemiluminescence
1° antibody
2° antibody
Protein-Protein Overlay Assay
4 membranes that are treated differently
 Membrane #1: CaCl2 (presence of Ca2+)
 Membrane #2: EGTA (absence of Ca2+)
 Membrane #3: control for buffer
 Membrane #4: control for antibodies

+ EGTA
Lane #:
GST 1
2
+ CaCl2
3 MW GST 1
2
3
MW
2
3
MW
1: 1.25 ug a-a
2: 2.5 ug a-a
3: 5 ug a-a
MW: molecular
weight standard
GST 1
2
3 MW
GST 1
GST: control for
antibody
+ nothing
no GST-CaM
(Control)
Protein-Protein Overlay
Enhanced
Chemiluminescence
1° antibody
2° antibody
Protein-Protein Overlay
Enhanced
Chemiluminescence
1° antibody
2° antibody
The Young et al hypothesis is supported.
1
218
269
Common domain:
the linker domain
749
Protein-Protein Overlay
Enhanced
Chemiluminescence
1° antibody
2° antibody
Protein-Protein Overlay
Enhanced
Chemiluminescence
1° antibody
2° antibody
The Tang et al hypothesis is supported.
1
269
Lacks the complete region of
Actin-binding domain, therefore
there is no sign of binding.
218
749
Problem encountered

Fragments of a-actinin in stock are:
CH1
GST
CH2
1
269
ABD
GST
1
2
3
218
4
749
GST
713
887
Problem encountered

If we do the protein-protein overlay, we
might expect the 1° antibody to recognize
the GST tag of a-actinin fragment.
CH1
GST
1
CH2
269
Problem encountered
Enhanced
Chemiluminescence
1° antibody
2° antibody
Thrombin CleanCleave Kit
Used Thrombin CleanCleave Kit to cleave the GSTtag off of the a-actinin fragments.
1
218
1
269
749
218
269
749
Products of the Cleavage Reaction
GST- a-actinin (1-269)
a-actinin (1-269) ~30 kDa
GST- ~27 kDa
Molecular
weight
Fragments of alpha actinin
CH1
1
218
CH2
1
2
3
4
3
2
269
4
1
CH2 CH1
713
749
1
887
887
Current progress

I am using the cleaved a-actinin fragments
to do more experiments.
Acknowledgement
Howard Hughes Medical Institute
Environmental Health Science Center
Dr. Jeffrey Greenwood
Dr. Kevin Ahern
Corey Singleton
Tamara Fraley
Thuan Tran
Scott Viner