Calcium regulation on Calcium regulation on actininactinin

byby

Cynthia SantosoCynthia Santoso

Dr. Jeffrey Greenwood LabDr. Jeffrey Greenwood Lab

Biochemistry and Biophysics DepartmentBiochemistry and Biophysics Department

CancerCancer

Cancer is a group of diseases Cancer is a group of diseases characterized by uncontrolled growth and characterized by uncontrolled growth and spread of abnormal cells.spread of abnormal cells.

If the spread is not controlled, it can result If the spread is not controlled, it can result in death.in death.

CancerCancer

Cancer is caused by both:Cancer is caused by both: external factors (tobacco, chemicals, external factors (tobacco, chemicals,

radiation, infectious organisms)radiation, infectious organisms) Internal factors (inherited mutations, Internal factors (inherited mutations,

hormones, immune conditions, and mutations hormones, immune conditions, and mutations that occur from metabolism)that occur from metabolism)

Who is at risk of developing Who is at risk of developing cancer?cancer?

ANYONE.ANYONE.

Since occurrence of cancer increases as Since occurrence of cancer increases as individuals age, most cases affect adults individuals age, most cases affect adults beginning in middle ages.beginning in middle ages.

CancerCancer

Types of cancer:Types of cancer:Benign tumorBenign tumor

- Remains confined to its original location, neither - Remains confined to its original location, neither invading surrounding normal tissue nor spreading invading surrounding normal tissue nor spreading to distant body sites.to distant body sites.

Malignant tumorMalignant tumor- Capable of both invading surrounding normal - Capable of both invading surrounding normal

tissue and spreading throughout the body via the tissue and spreading throughout the body via the circulatory or lymphatic system.circulatory or lymphatic system.

CancerCancer

Only malignant tumors are properly Only malignant tumors are properly referred as cancers, and it is the ability to referred as cancers, and it is the ability to invade and metastasize that makes cancer invade and metastasize that makes cancer so dangerous.so dangerous.

* metastasis refers to the spread of cancer * metastasis refers to the spread of cancer cells from their site of origin to other sites cells from their site of origin to other sites in the body.in the body.

MetastasisMetastasis

• The very important process by which cancer spreads from one part of the body to another.

• How do these cancer cells move about?

MetastasisMetastasis Moving cells, whether healthy or Moving cells, whether healthy or

cancerous, grab the extracellular cancerous, grab the extracellular matrix with tiny feet, called focal matrix with tiny feet, called focal adhesion complexes. adhesion complexes.

They move their way up along the They move their way up along the extracellular matrix.extracellular matrix.

Upon reaching a vein or artery, Upon reaching a vein or artery, they have a long trip throughout they have a long trip throughout the body.the body.

At the end of the journey, they exit At the end of the journey, they exit the blood vessel and found a new the blood vessel and found a new location to further grow.location to further grow.

Cell AdherenceCell Adherence

Cell adhesion is an important mechanism by which cells Cell adhesion is an important mechanism by which cells interact with the extracellular environment.interact with the extracellular environment.

The adhesive state of a cell has significant influence on The adhesive state of a cell has significant influence on growth and survival, migration, and signal transduction.growth and survival, migration, and signal transduction.

AttachedCell

SpreadCell

Cell with stress fibersand focal adhesions

WeakAdherence

IntermediateAdherence

StrongAdherence

De-adhesion

Focal Adhesion StructureFocal Adhesion Structure

Talin

Vinculin

Paxillin Talin TalinTalin -actinin -actinin

-actinin

-actinin-actinin-actinin

-actinin-actinin

-actinin

FAK

Syndecan-4

Extracellular Matrix

Strong Adherence -cell with stress fibersand focal adhesions

IntermediateAdherence

PI 3-kinasePDGF

P T

T

P

T

-a

-a-a

T

-a-a

-a

VV

V V

-a -a

= PtdIns (3,4,5)-P3

PDGF treated fibroblasts

Hallmark of cell adhesion and Hallmark of cell adhesion and motility studiesmotility studies

It enables us to modulate or adjust or vary It enables us to modulate or adjust or vary these signals in order to control both these signals in order to control both desirable and undesirable cell growth and desirable and undesirable cell growth and motility.motility.

Alpha-actinin structureAlpha-actinin structure

-actinin is an anti-parallel homodimer.-actinin is an anti-parallel homodimer. Each monomer is composed of three domains: Each monomer is composed of three domains:

The actin-binding domainThe actin-binding domain The spectrin repeatsThe spectrin repeats The C-terminal EF hands domainThe C-terminal EF hands domain

ABDSpectrin repeatsEF hands

4321

1234 CH1CH2

CH2CH1

1 256 364 479 600 713 887141

FactFact

• It has been known that calcium regulates It has been known that calcium regulates actinin bundling activity.actinin bundling activity.

• ““Calcium oscillation trigger focal adhesion Calcium oscillation trigger focal adhesion disassembly…” by Giannone et al.disassembly…” by Giannone et al.

QuestionQuestion

1. Do the EF Hands domains bind 1. Do the EF Hands domains bind specifically to the Actin-binding domains?specifically to the Actin-binding domains?

2. If so, is the binding regulated by Calcium 2. If so, is the binding regulated by Calcium ion?ion?

The Young et al HypothesisThe Young et al Hypothesis

The closed or inactive state of the The closed or inactive state of the molecule exists when the EF 3/4 region of molecule exists when the EF 3/4 region of the CaM-like region of the CaM-like the CaM-like region of the CaM-like domain interacts with a region between domain interacts with a region between the ABD and R1 of the opposite subunit.the ABD and R1 of the opposite subunit.

Ca2+

Ca2+

The Tang et al HypothesisThe Tang et al Hypothesis In the presence of Ca2+, the CAL domain (EF In the presence of Ca2+, the CAL domain (EF

Hands) of Hands) of -actinin could undergo a -actinin could undergo a conformational change that enables it to wrap conformational change that enables it to wrap around… the connecting helix between the around… the connecting helix between the two CH domains in the ABD.two CH domains in the ABD.

Ca2+

Ca2+

MethodMethod

Protein-protein overlay assay.Protein-protein overlay assay.We did overlay with the full length of We did overlay with the full length of --

actinin.actinin.

Protein-Protein Overlay AssayProtein-Protein Overlay Assay

1° antibody 2° antibody

Enhanced Chemiluminescence

Protein-Protein Overlay AssayProtein-Protein Overlay Assay

4 membranes that are treated differently4 membranes that are treated differentlyMembrane #1: CaClMembrane #1: CaCl22 (presence of Ca (presence of Ca2+2+))Membrane #2: EGTA (absence of CaMembrane #2: EGTA (absence of Ca2+2+))Membrane #3: control for bufferMembrane #3: control for bufferMembrane #4: control for antibodiesMembrane #4: control for antibodies

GST 1 2 3 MW

GST 1 2 3 MW GST 1 2 3 MW

GST 1 2 3 MW

+ EGTA + CaCl2

+ nothing no GST-CaM (Control)

Lane #:

1: 1.25 ug -a

2: 2.5 ug -a

3: 5 ug -a

MW: molecular weight standard

GST: control for antibody

Protein-Protein OverlayProtein-Protein Overlay

1° antibody 2° antibody

Enhanced Chemiluminescence

1° antibody 2° antibody

Enhanced Chemiluminescence

Protein-Protein OverlayProtein-Protein Overlay

The Young et al hypothesis is supported.The Young et al hypothesis is supported.

1 269

218 749

Common domain: the linker domain

Protein-Protein OverlayProtein-Protein Overlay

1° antibody 2° antibody

Enhanced Chemiluminescence

1° antibody 2° antibody

Enhanced Chemiluminescence

Protein-Protein OverlayProtein-Protein Overlay

The Tang et al hypothesis is supported.The Tang et al hypothesis is supported.

1 269

218 749

Lacks the complete region of Actin-binding domain, therefore

there is no sign of binding.

Problem encounteredProblem encounteredFragments of Fragments of -actinin in stock are:-actinin in stock are:

GST

713 887

GST

1 269

CH2CH1

ABD

218

GST

749

4321

Problem encounteredProblem encountered

If we do the protein-protein overlay, we If we do the protein-protein overlay, we might expect the might expect the 1° antibody to recognize the GST tag of actinin fragment.

GST

1 269

CH2CH1

Problem encounteredProblem encountered

1° antibody 2° antibody

Enhanced Chemiluminescence

1 269

218 749

1 269

218 749

Thrombin CleanCleave KitThrombin CleanCleave Kit

Used Thrombin CleanCleave Kit to cleave the GST- tag off of the -actinin fragments.-actinin fragments.

Products of the Cleavage ReactionProducts of the Cleavage Reaction

GST- -actinin (1-269)

-actinin (1-269) ~30 kDa

GST- ~27 kDa

Molecular weight

Fragments of alpha actininFragments of alpha actinin

4321

1234 CH1CH2

CH2CH1

1 269

218 749 1 887

713 887

Current progressCurrent progress

I am using the cleaved I am using the cleaved -actinin fragments -actinin fragments to do more experiments.to do more experiments.

AcknowledgementAcknowledgement

Howard Hughes Medical InstituteHoward Hughes Medical InstituteEnvironmental Health Science CenterEnvironmental Health Science Center

Dr. Jeffrey GreenwoodDr. Jeffrey GreenwoodDr. Kevin AhernDr. Kevin AhernCorey Singleton Corey Singleton Tamara FraleyTamara Fraley

Thuan TranThuan TranScott VinerScott Viner