Question

1. You are studying an integral plasma membrane protein named LINKER that can interact with the cytoskeletal molecule spectrin to form complex matrices across the surface of membranes. To begin to understand how the LINKER protein population responds to spectrin availability, you perform some fluorescence recovery after photobleaching (FRAP) experiments using a LINKER-GFP fusion protein. The results of a FRAP experiment with the wildtype version of LINKER in which 50% of the protein population is bound to spectrin is shown in the graphs on the left below. Next, you make two modifications: A) you generate a mutant form of LINKER-GFP that can no longer bind to spectrin and B) you express wildtype LINKER-GFP in a naturally occurring cell line variant that dramatically overexpresses spectrin. Diagram the expected FRAP results in these two situations on the diagrams on the right below. Provide a short explanation for each of your predictions. (3 points each, 6 points total) photobleach photobleach 100 - A) 1001_ 0 WT 5 time (min) 10 10 time (min) photobleach photobleach 100 " WT 0 10 10 5 time (min) 5 time (min) 1C. A rise in intracellular Ca2+ concentration causes muscle cells to contract. In addition to an ATP- driven Ca?* pump, heart muscle cells, which contract quickly and regularly, have an antiporter that exchanges Ca?* for extracellular Na* across the plasma membrane. This antiporter rapidly pumps most of the entering Ca2+ ions back out of the cell, allowing the cell to relax. You are working a lab that has identified a putative new drug for the treatment of heart disease called C-TRCT because it makes heart muscle cells in a mouse model for heart disease contract more strongly. While studying the physiological effect of C-TRCT on mouse heart muscle cells, you find that it partially inhibits the Na*-* pump in the membrane of heart muscle cells. Given this information, provide an explanation for how C- TRCT leads to stronger muscle contraction in heart cells. (3 points)

Answer 1

Spectrin is an intracellular protein that is part of the cytoskeletal structure of a cell. It forms a scaffold structure in the inner side of the plasma membrane, aiding in the maintenance of membrane structure and integrity. FRAP, or Fluorescence Recovery After Photobleaching, is the process which involves bleaching of fluorophores (like GFP) attached to the extracellular end of membrane proteins, by the use of intense laser light. As a result of the bleaching of fluorophores on the cell membrane, there is a loss of fluorescence on the cell surface. However, the plasma membrane is known to be fluid (not static) where the proteins and lipids are free to move along the length of the membrane. As a result of this, the proteins which had their fluorophores bleached gradually mix with proteins which still have their fluorophores intact, thereby decreasing the average loss in fluorescence in the bleached area. This procedure is called Fluorescence Recovery After Photobleaching, or FRAP.

In this problem it has been stated that the Linker protein is an integral membran protein which interacts with spectrin to form matrices across the membrane surface. It has also been observed that in wild-type Linker protein population, 50% of the Linker proteins are attached to spectrin on the intracellular face of the membrane. Since spectrin is a cytoskeletal protein, therefore it is not possible for spectrin to move around along the inner membrane surface. Spectrin participates in the maintenance of the cell membrane structure ; therefore, it is imperative that the position of spectrin be fixed. From this fact it can be inferred that the Linker proteins attached to the spectrin would also not be free to move across the membrane, which they otherwise would have (without spectrin interaction). Therefore, in the given problem, 50% of the Linker proteins are attached while the rest 50% are free to move across after FRAP, hence producing the graph. Now, two additional experimental states have been mentioned involving the Linker protein and spectrin, which would likely yield results different from the one we obtained using wild-type Linker protein.

A. In this case, a mutant form of Linker protein is being expressed by the cell, which is unable to interact with spectrin, This Linker protein too, like its wild type version, has a GFP tag fused to it, in order to study fluorescence activity. Since this mutant Linker protein is not able to bind to spectrin, therefore in this case all the Linker proteins are free to flow across the membrane and diffuse into the bleached area. Therefore, the population of Linker proteins aiding in fluorescence recovery are much higher in this case, which would lead to an increase in fluorescene recovery as compared to the graph with wild-type Linker.

mutant linker a wild- Type linker fludescence I Recovery 10 . CS) Scanned with CamScanner

B. In this experiment, the wild-type Linker protein is fused to a GFP and expressed in a cell line which greatly overexpresses spectrin. As a result of the increased number of spectrin molecules in the intracellular surface, more number of Linker proteins will be involved in interacting with spectrin. As a result, a greater number of Linker proteins will be bound to fixed positions (due to spectrin interaction) and fewer Linker molecules would be left to diffuse across the membrane to the bleached area. Consequently, fluorescence recovery would suffer and the fluorescence would be much lesser than the one observed with wild-type Linker and normal spectrin levels.

. Į fluorescence :- Normal Specfrin expressin High Spectrin Enpresion immary) > 10 Scanned with CamScanner

1C. The function of the Na+/K+ ATPase is to maintain a low concentration of sodium (Na+) inside the cell and a high intracellular concentration of potassium (K+). In case of the inhibition of thie Na+/K+ ATPase pump in heart muscle cells , there is a rise in the intracellular concentration of Na+. An increase in intracellular sodium concentration leads to an increase in Ca2+ concentration inside the cell, via the Na+-Ca2+ antiporter, as stated in the question. The disease C-TRCT has been proven to inhibit the Na+/K+ ATPase pump in heart cells. Therefore, in patients affected by C-TRCT, the inhibition of the sodium-potassium pump leads to an accumulation of sodium inside the cell, which in increases the concentration of calcium inside the cell. The rise in intracellular calcium (Ca2+) casues the contraction of the myocardial cells or heart muscel cells. This is how incidence of C-TRCT brings about strong contraction of cardiac muscle cells.