Cycle 1: Chlamydomonas and how it uses light

• Prokaryotic cells have..

  - No cell nucleus.

  - No membrane encased organelles.

  - Capsule.

  - Cell wall (In some eukaryotes).

  - Flagellum.

  - Tend to be smaller.
• Eukaryote cells have..

  - Possess membrane bound organelles.

  - Membrane-bound nucleus that holds genetic material.

  - Chloroplast, mitochondria.

  - Nuclear membrane.

  - Golgi.
• prokaryotes and eukaryotes both have...

  Cell membrane, ribosomes, cell wall (in some eukaryotes).
• Why is chlamydomonas a good model system?

  - Has an eyespot.

  - Can be grown in the lab, grow different strains, using agar plates.

  - Good model to study a number of human diseases that collectively are called ciliopathies
• Chlamydomonas life cycle

  In favourable conditions:

  - Moderate temperature, moderate light, lots of nitrogen, nutrients, allows cells to divide.

  Unfavourable conditions:

  - Switch to sexual reproduction. Why would they switch (from asexual to sexual)? Zoospores in asexual production are haploid, zygotes in sexual reproduction are diploid. in sexual reproduction, more variations are produced. Thus, it ensures the survival of species in a population. The newly formed individual has characteristics of both the parents. Variations are more viable in sexual mode than in asexual one.

  - Nutrients are lower, dry environment.
• open reading frame (ORF)


  Predication about how many protein coding genes a piece of DNA have.
• When generation time is increased, ..

  Complexity of the organism is also increased.
• Is photosynthesis just found in eukaryotes?

  No. Prokaryotic photosynthetic organisms have in-foldings of the plasma membrane for chlorophyll attachment and photosynthesis. It is here that organisms like cyanobacteria can carry out photosynthesis. Some prokaryotes can perform photosynthesis.
• Structure of flagellum is virtually identical between Chlamydomonas and humans. How is that?


  They share a common ancestor. Flagella was simply lost from plant lineage.
• Ciliopathies


  Human diseases linked to defects in cilia, e.g. polycystic kidney disease
• What would be an example of a protein that all three (Chlamydomonas, humans, arabidopsis) would have?


  Enzymes (a guess).
• Arabidopsis


  Tiny weed that is often used for plant research, because it is very easy to grow, and its DNA has been mapped.
• Phototaxis


  movement in response to light
• positive phototaxis


  movement towards light
• negative phototaxis


  movement away from light
• Why would wild type cells display negative phototaxis?

  Phototaxis is advantageous for phototrophic organisms as they can orient themselves most efficiently to receive light for photosynthesis.
• What is the function of an eyespot in Chlamydomonas?

  Vision evolved in motile, single-celled, green algae to enhance photosynthetic capability. A specialized structure within the cell, the eyespot, aids in the detection of light direction and is key to improving the efficiency of phototactic behaviour.
• phototactic response


  movement toward or away from light
• Can you think of a gene where if that gene was defective it would prevent phototaxis?

  Any gene that is essential to the function of the eyespot, which is what causes the phototactic response, would prevent phototaxis.
• Eyespot


  light sensor, allows Chlamydomonas to gather information about the direction and intensity of the light in its environment. The eyespot is around the mid-axis of a Chlamydomonas cell.
• Chlamydomonas


  Unicellular green algae that undergo both sexual and asexual reproduction.

  Characteristics:- Single-celled, photosynthetic eukaryote- Commonly found in ponds and lakes- Contains a single large chloroplast that harvests light energy and uses it to make energy-rich molecules through the process of photosynthesis.- Each cell contains a light sensor called an eyespot that allows Chlamydomonas to gather information about the direction and intensity of the light in its environment.

  
• Overall structure of the eyespot has two major facets that are important:


  Front side:Eyespot needs to absorb light. When light comes in at the front, the photon can impinge directly and be absorbed by the channelrhodopsin molecule. Or it could miss and it would bounce off one of these carotenoid layers, come back and then be absorbed by channelrhodopsin on the rebound. Carotenoid acts as a reflector, which increases the amount of photons that get absorbed by channelrhodopsin.

  Backside:eyespot is blind to light coming in from the rear side. If light comes in from the back it never gets to channelrhodopsin. The carotenoid layer acts as a barricade that blocks the photons from reaching the Channelrhodopsin and being absorbed.This is important because it enables a Chlamydomonas cell to know whether its in the dark or the light when it spins around. It knows the direction of light, so that it can phototactic, move towards or away from the light.

  
• Channelrhodopsin

  A protein complex, light-gated ion channel. Light gate meaning the gate is closed, then this is able to interact with light, absorption of a photon of light causes the gate to open, and then it'll close down again.
• How does channelrhodopsin work?

  Plasma membrane of every eukaryote has a voltage across it. Inside of plasma membrane is always negatively charged. There's a voltage difference across this plasma membrane.

  
  

  When Channelrhodopsin interacts with light, the gate opens, and it allows very specific cations to move through into the cell. Specifically calcium, and protons. It's a pore. When the channel opens up, it enables these ions to move down the electrochemical gradient. They're simply attracted to the negative charge inside.

  
• membrane potential

  The voltage across a cell's plasma membrane. There's a strong voltage gradient across the membrane. When Channelrhodopsin opens up and allows these ions through, what's the effect? You're depolarizing the membrane. Now the inside is not quite as negatively charged as it used to be.

  
  

  - The membrane potential difference across the membrane is usually pretty high.

  - When the gate opens up, it causes a spike, this depolarizing, then it will shut back down and become repolarized again.

  - Strongly polarized membrane, depolarizes, then repolarizes.

  - This is an action potential on the plasma membrane.

  - Exact same way a neuron fires. You have a threshold potential, once you've exceeded that, this action potential is triggered when Channelrhodopsin opens up.

  
• Action potential & phototaxis relationship


  We have the eyespot absorbing a photon of light, the action potential moves from the eyespot down to the base of theflagella. In ways we don't understand, those action potentials that fire and migrate down, are interpreted by the base of the flagella and that information causes that flagella to move, and theChlamydomonas to swim.
• Opsin


  a class of protein that, together with retinal, constitutes the photopigments

  
• Channelrhodopsin is made out of two components..

  The protein opsin, and the opsin binds this pigment called retinal.

  
  

  Channelrhodopsin is a photoreceptor. Light-gated ion channel made up of retinal and opsin, and its also termed a photoreceptor.
• Retinal absorbs strongly in

  blue light.

  
• absorption spectrum


  The range of a pigment's ability to absorb various wavelengths of light.
• What's absorption?

  The energy of the photon, that photon of light is used to excite an electron from a ground state to an excited state. That's the definition of light absorption.
• Chlorophyll can absorb...

  Red as well as blue light. Because blue light is more energetic, shorter wavelength, more energy per photon, then it can excite that same electron to ahigher excited state.

  For chlorophyll to absorb a photon of light, the energy of the photon must match perfectly the energy required to get an electron from the ground state to the excited state.Ex. The fact that this electron can absorb a red photon, means that the energy of a red photon matches perfectly this energy gap. Same with blue photons, since chlorophyll can absorb both.
• Why does chlorophyll look green?

  Chlorophyll looks green because it can't absorb the green photons of light. The green photons of light get bounced off or passed through, which is why you end up seeing it.
• Where does chlorophyll's energy come from?

  The lower excited state electron is the electron that ends up going down electron transport and driving those reactions that fuel the Calvin cycle, etc. The red excited state is what fuels the biosphere.
• Why can't chlorophyll use the higher blue excited state?


  Its relatively unstable. Before anything can happen, before the photosynthetic machinery can actually use this excited state here, the excited state decays, heat loss, and turns into a lower excited state. Decay down to the red excited state.
• photoisomerization

  a change in shape by a photopigment molecule from one isomer (11-cis retinal) to another (all-trans retinal) when the molecule absorbs a photon; initiates the transduction of light to a neural signal.When channelrhodopsin absorbs a photon of light and excites an electron, its causing photoisomerization.
• Molecule of retinal during photoisomerization

  If we look at the molecule of retinal, what's happening upon photon absorption is that you're going from trans retinal to cis. When the blue photon gets captured by the electron, it goes to the higher excited state, for a split second this double bond is broken because one electron is in a higher excited state, and it enables the tail end of this molecule to swivel around what is now essentially a single bond, then the electron that is in the excited state decays back down to the ground state and the double bond reforms, in the cis conformation, and you're left with a cis formation instead of trans.

  In the cis conformation, its no longer linear, there's a kink on the end.
• What drives the conformational change of opsin?


  Opsin changes conformation, and this change in conformation is pore opening.

  
  

  The retinal molecule is bound very precisely in a pocket, when it absorbs a photon of light, the photoisomerization that results causes a kink a change in the conformation of retinal that drives a conformational change in the opsin protein. It shifts from being a closed gate to being open.

  
• Photoreceptor molecules in a rod cell..

  The photoreceptor molecules in a rod cell are found in the photoreceptor discs.

  
  

  If we look closely at the photoreceptor discs. Inside the discs there are photoreceptors, the photoreceptors are the proteins opsin and they bind the pigment retinal. Together, they produce the photoreceptor rhodopsin.
• Rhodopsin

  The photoreceptor in your eye is called rhodopsin. Its comprised of the protein opsin, plus the pigment retinal. (Square blue and green boxes on disk). When light hits rhodopsin, retinal changes shape. You cause the change in shape in opsin the exact same way as in channelrhodopsin. Unlike channelrhodopsin, rhodopsin is not a light gated channel. It doesn't open to allow ions to go through.The change in shape triggers a signal transduction pathway.