Calcium regulation on   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. 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. If the spread is not controlled, it can result in death.

Cancer Cancer is caused by both: Cancer is caused by both: external factors (tobacco, chemicals, radiation, infectious organisms) external factors (tobacco, chemicals, radiation, infectious organisms) Internal factors (inherited mutations, hormones, immune conditions, and mutations that occur from metabolism) Internal factors (inherited mutations, hormones, immune conditions, and mutations that occur from metabolism)

Who is at risk of developing cancer? ANYONE. ANYONE. Since occurrence of cancer increases as individuals age, most cases affect adults beginning in middle ages. Since occurrence of cancer increases as individuals age, most cases affect adults beginning in middle ages.

Cancer Types of cancer: Types of cancer: Benign tumor Benign tumor - Remains confined to its original location, neither invading surrounding normal tissue nor spreading to distant body sites. Malignant tumor 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. 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. 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. They move their way up along the extracellular matrix. Upon reaching a vein or artery, they have a long trip throughout the body. 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. At the end of the journey, they exit the blood vessel and found a new location to further grow.

Cell Adherence Cell adhesion is an important mechanism by which cells interact with the extracellular environment. 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. The adhesive state of a cell has significant influence on growth and survival, migration, and signal transduction. Attached Cell Spread Cell Cell with stress fibers and focal adhesions Weak Adherence Intermediate Adherence Strong Adherence De-adhesion

Focal Adhesion Structure           Talin Vinculin   Paxillin Talin  -actinin FAK Syndecan-4 Extracellular Matrix

Strong Adherence - cell with stress fibers and focal adhesions Intermediate Adherence PI 3-kinasePDGF       P T   T   P T    -a T V V     V V = PtdIns (3,4,5)-P 3 PDGF treated fibroblasts

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. 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  -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 domain The actin-binding domain The spectrin repeats The spectrin repeats The C-terminal EF hands domain The C-terminal EF hands domain ABD Spectrin repeats EF hands CH1 CH2 CH

Fact It has been known that calcium regulates  actinin bundling activity. It has been known that calcium regulates  actinin bundling activity. “Calcium oscillation trigger focal adhesion disassembly…” by Giannone et al. “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? 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. 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+

The Tang et al Hypothesis In the presence of Ca2+, the CAL domain (EF Hands) of  -actinin could undergo a conformational change that enables it to wrap around… the connecting helix between the two CH domains in the ABD. In the presence of Ca2+, the CAL domain (EF Hands) of  -actinin could undergo a conformational change that enables it to wrap around… the connecting helix between the two CH domains in the ABD. Ca 2+

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

Protein-Protein Overlay Assay 1° antibody2° antibody Enhanced Chemiluminescence

Protein-Protein Overlay Assay 4 membranes that are treated differently 4 membranes that are treated differently Membrane #1: CaCl 2 (presence of Ca 2+ ) Membrane #1: CaCl 2 (presence of Ca 2+ ) Membrane #2: EGTA (absence of Ca 2+ ) Membrane #2: EGTA (absence of Ca 2+ ) Membrane #3: control for buffer Membrane #3: control for buffer Membrane #4: control for antibodies Membrane #4: control for antibodies

GST MW + EGTA+ CaCl2 + nothingno 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 Overlay 1° antibody2° antibody Enhanced Chemiluminescence

1° antibody2° antibody Enhanced Chemiluminescence Protein-Protein Overlay

The Young et al hypothesis is supported Common domain: the linker domain

Protein-Protein Overlay 1° antibody2° antibody Enhanced Chemiluminescence

1° antibody2° antibody Enhanced Chemiluminescence Protein-Protein Overlay

The Tang et al hypothesis is supported Lacks the complete region of Actin-binding domain, therefore there is no sign of binding.

Problem encountered Fragments of  -actinin in stock are: Fragments of  -actinin in stock are: GST GST 1269 CH2 CH1 ABD 218 GST

Problem encountered If we do the protein-protein overlay, we might expect the If we do the protein-protein overlay, we might expect the 1° antibody to recognize the GST tag of  actinin fragment. GST 1269 CH2 CH1

Problem encountered 1° antibody2° antibody Enhanced Chemiluminescence

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

Products of the Cleavage Reaction  GST-  -actinin (1-269)   -actinin (1-269) ~30 kDa GST- ~27 kDa Molecular weight

Fragments of alpha actinin CH1 CH2 CH

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