Teacher PD Days 2014 Some interesting aspects of cellular processes

Mitosis

Cell Structures and Cell Division Videos K2w&list=UUGJqRYuHwar5W6fzQO4VxxQ K2w&list=UUGJqRYuHwar5W6fzQO4VxxQ ZXo ZXo Pg84 Pg84 ip0

Immunostaining. It is relatively easy to make antibodies that specifically recognise only one protein and link this to a fluorescent chemical. Then we can add this to a cell that has been “fixed” using chemical to make it rigid and permeable We can then visualise using a fluorescent microscope. The good thing about fluoresent compounds is that the light emits over a very narrow range of wavelength which means we can monitor more that one protein at a time by use more than one antibody and using different fluorescent probes Two major methods used to let us know what is really going on with proteins inside the cell Microtubules and nucleiActin and nuclei Golgi and nuclei

Two major methods used to let us know what is really going on with proteins inside the cell (Green) Fluorescent Protein. These can be genetically engineered for a contiguous fusion protein with pretty much any protein and then this engineered gene can be reintroduced into cells so the the fluorescent protein part can act a a marker to track the other Basic structure now engineered to make proteins that fluoresce at a wide range of wavelengths

Kremers G et al. J Cell Sci 2011;124: ©2011 by The Company of Biologists Ltd

Dual Immunostaining of GLUT4 and a Protein It Associates With DOI: /sj.emboj |Published

Movie of GLUT4-GFP fusion moving to the plasma membrane after insulin in adipocytes stimulated with insulin For the aficianados this was done using a technique called TIRF microscopy that only looks at the plasma membrane so this is measuring GLUT4 coming to the plasma membrane. From Mol. Cell. Biol 2007 vol 27 p 3456

Respiration Videos 0Y g Y Y

Cellular Energy Metabolism and Respiration We mainly think of glycolysis and Krebs cycle (i.e respiration) as a way to use glucose to producea lot of energy in the form of ATP BUT don’t forget its also the ways we produce all our heat. Also don’t forget we can use other sugars, fats and amino acids to produce energy in the form of ATP using these same pathways. The two major linked mechanisms for achieving this are Glycolysis and Krebs Cycle Glycolysis happens in cytosol and can proceed without oxygen but only produces 2 ATPs for each glucose used Krebs cycle in mitochondria greatly amplifies this response by taking end point from glycolysis and generating up to another 34 ATP from each glucose in a process that uses O 2 and produces CO 2 plus produces heat.

Metabolism of Glucose KREBS CYCLE

Metabolism of Other Nutrients Lactose Galactose

Metabolism of Other Nutrients Sucrose Fructose Fructose-1- Phosphate

Metabolism of Other Nutrients Triglycerides Glycerol Fatty Acids Acetyl CoA KREBS CYCLE

Metabolism of Other Nutrients Alanine CH 3 C COOH NH 2 H Alanine CH 3 C COOH O Pyruvate Aminotransferase

Acetyl CoA (2carbons) C C C C C C C C C C C C Citrate (6 carbons) Oxaloacetate (4 carbons) Citrate (5carbons) NADH e- CO 2 C C NADH e- CO 2 C C Succinyl CoA (4 carbons) FADH 2 e- NADH e- C C C C C C C C ATP C C C C C C C C C C Electron transport chain linked to use of oxygen and heat production ATP The start of the Krebs cycle in the mitochondria, a good example of how organelles allow the cell to perform specialist roles by have a separate environment Into mitochondria from cytosol Acetyl CoA Pyruvate from glycolysis C C C C C C C C C C C C

Acetyl CoA (2carbons) C C C C C C C C C C C C Citrate (6 carbons) Oxaloacetate (4 carbons) Citrate (5carbons) NADH e- CO 2 C C NADH e- CO 2 C C Succinyl CoA (4 carbons) FADH 2 e- NADH e- C C C C C C C C ATP C C C C C C C C C C The bit where the CO 2 comes off C C C C C C C C C C C C

Acetyl CoA (2carbons) C C C C C C C C C C C C Citrate (6 carbons) Oxaloacetate (4 carbons) Citrate (5carbons) NADH e- CO 2 C C NADH e- CO 2 C C Succinyl CoA (4 carbons) FADH 2 e- NADH e- C C C C C C C C ATP C C C C C C C C C C C C C C C C C C C C C C

Acetyl CoA (2carbons) C C C C C C C C C C C C Citrate (6 carbons) Oxaloacetate (4 carbons) Citrate (5carbons) NADH e- CO 2 C C NADH e- CO 2 C C Succinyl CoA (4 carbons) FADH 2 e- NADH e- C C C C C C C C ATP C C C C C C C C C C Electron transport chain linked to use of oxygen and heat production ATP

Final step happens in the inner mitochondrial membrane

Respiration factoids We have all heard about how oxidative damage can be bad for us and anti-oxidants are good for us – how does this happen. There is a lot of electron transport going on at the mitochondrial membrane so sometimes the wrong molecules get the electrons to make highly reactive molecules likes hydrogen peroxide and oxygen free radicals. These can oxidise all sorts of biological molecules and often this can cause damage. Antioxidants are supposed to mop up these oxidative species.

Final step happens in the inner mitochondrial membrane ATP Synthase

Final step happens in the inner mitochondrial membrane ATP Synthase CoupledCoupled CoupledCoupled Heat production and O 2 can not normally proceed without having to also produce ATP

Final step happens in the inner mitochondrial membrane Now heat production and O 2 use can proceed without having to also produce ATP Uncoupling protein

Respiration facts Normally the electron transport chain is tightly coupled to ATP levels so if we have too much ATP then the complex making the ATP stops working. Since the only way for the H+ ions to get back across the membranes fast enough is via the ATPase making ATP then the gradient of H+ ions builds up and then the electron transport slows down (there is no need to transport H+ ions) so we use less O 2 and we use less NADH so we use less nutrient. Therefore we say that respiration is “coupled” to the use of ATP. This is to make sure we don’t keep burning energy unless we need to. However, some mitochondria have a protein in the inner membrane that lets H+ ions move freely back across the membrane. These are called “Uncoupling proteins” because they decouple the production of ATP from the rest of the electron transport chain. Why would a cell want to do that ? The reason is that this means the electrons can still be transported to oxygen to generate heat. This means we can use up NADH and so nutrients to produce heat but not ATP. This is pretty useful if you are a hibernating bear for example.

Respiration factoids Factoid - Cyanide works by blocking electron transport chain. We can produce extra heat by muscle use so body uses this to keep warm by shivering so heat production that doesn’t use this method (e.g. in brown fat with uncoupling protein) is called non-shivering thermogenesis). Factoid – Brown fat is brown because it has so many mitochondria. The reason mitochondria are is because the electron transport chain molecules have so much iron in the heme prosthetic groups that transport the electrons