Cycle 1: Chlamydomonas and how it uses light Flashcards

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1
Q

Major structural features of a Chlamydomonas cell (we will fill these in over the next few cycles).

A

Chlamy is a eukaryotic green algae:

  • About 10microns across
  • Possess chloroplasts
  • 2 flagella that enable motion and can be regrown using the basal body
  • Eyespot, partially in chloroplast and partially on membrane, that senses the light environment to indicate to flagella which direction to move in
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2
Q

Features of Chlamy grown in the lab.

A
  • Usually grown as haploid cells, only 1 copy of every chromosome
  • Under moderate light and temperature (24-28deg)
  • GROWS by binary fission (with cell division occuring every ~10h if optimum condition)
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3
Q

Growth media…what is the difference between a Macronutrient and a Micronutrient.

A

Chlamy growth media is liquid media: TAP, with various essential salts

Macronutrient: nutrients required in larger amounts by organisms

Micronutrient: nutrients required in smaller amounts by organisms

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4
Q

Look up why Chlamy (and humans) need PO4 (phosphate) and Fe (iron).

A

Phosphate required for DNA sugar-phosphate backbone, in phospholipid cell bilayer, forms ATP in cellular respiration/photosynthesis.
Iron required to bind to hemoglobin as a cofactor and transport oxygen.*

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5
Q

Chlamy grows with a doubling time of about 10 hours at 25C….what does that actually mean…grows and doubling time?

A

Chlamy’s DOUBLING/GENERATION TIME refers to the time required for a colony to double in number (for enough binary fission to occur that each cell splits in two) - occurs during exponential growth phase.

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6
Q

Microbial growth curve….what explains its shape….three major phases…(Lag, Exponential, Stationary) what’s going on in each.

A

Graph - Cells/mL as a function of time

LAG - shallow slope, slow growth rate (cells adjust, synthesize enzymes, prep for growth)
EXPONENTIAL - steep slope, doubling time phase (high growth rate, exponential increase in cell number) (abundant nutrients, favourable conditions for reproduction)
STATIONARY - plateau slope, growth slows again (nutrients depleted, waste accumulated, cell growth equals cell death)

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7
Q

Bacteria have flagella too…but nothing like eukaryotic flagella….analagous structures.

A

Bacteria (prokaryotes) have flagella too but they are structurally completely different from eukaryotic flagella (they spin instead of waving like in eukaryotes, they have filament and hooks instead of microtubules), because they evolved in parallel - the flagella are ANALOGOUS! (Analogs refer to traits that are similar but evolved independently as a response to a similar challenge. Homologs refer to traits that are similary because they share a common ancestry.)

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8
Q

Chlamydomonas phylogeny….relationship to plants, animals.

A

Chlamy (and all algae) are photosynthetic eukaryotes but are not plants! They share a common ancestor with plants a billion years ago.

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9
Q

How can we explain that Chlamydomonas and humans have flagella but plants do not. What is the simplest explanation?

A

Chlamy and humans have flagella because they share a common ancestor, however, plants lost the ability to have flagella during evolution because it wasn’t evolutionarily advantageous to keep them.

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10
Q

Basics of cilia structure (proteins involved, structures)

A

Flagella and cilia are the same thing!
- Contain microtubules (protein polymer (hollow tube) made from alpha and beta tubulin dimers)
- Flagella have 9 microtubule doublets around and 2 central microtubules
- Dynein arms (motor protein) which move along one side of adjacent doublets (from - to + end) causing flagella to move in a whip-like fashion

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11
Q

Mutations that alter cilia structure/function cause many human disease states (ciliopathies) (recall, we make no distinction between cilia and flagella).

A

Ciliopathies are distinct diseases linked to mutations in genes involved in cilia structure/function. If dynein is missing (MUTATION IN DYNEIN SYNTHESIS), all cilia become non-motile even if they’re supposed to be motile.

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12
Q

Distinctions between motile and non-motile (sensory) flagella. You do not need to memorize that figure with all the diseases!

A

Motile cilia (lungs, intestinal tract) move and non-motile cilia (ears, eyes, nose) do not move.

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13
Q

Ciliopathies are often genetically heterogenous…what does that mean?

A

The same ciliopathies can be caused by mutations in more than one gene, since more than one gene codes for microtubules. It is said to be heterogenous because the same phenotype can be caused by mutations in more than one gene.

Proteins required for flagella can be isolated using SDS-PAGE to compare WT and bbs4 proteins (have no flagella, incapable of phototaxis).

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14
Q

Analysis of 7,476 Chlamy proteins…comparision with proteins in humans, arabidopsis and both those species.

A
  • 26% are only homologs to arabidopsis (1968 Chlamy proteins that are evolutionarily related to 2396 plant proteins), likely related to photosynthesis, etc.
  • 10% are only homologs to humans (774 Chlamy proteins ~ 806 human proteins), likely related to flagella, etc.
  • 33% are homologs to both arabidopsis and humans, likely related to general eukaryotic function?
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15
Q

What characteristics does Chlamy have that makes it a “model experimental system”? [IMPORTANT]

A
  • Chlamy’s flagella is identical to human cilia and has the same proteins SO it is used to study ciliopathies
  • Chlamy is easier/less expensive to study than human cells
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16
Q

What is the reason Chamy stops dividing during stationary phase?

A

*

17
Q

Analysis of 7,476 Chlamy proteins: What structures/processes (and thus proteins/genes) are common to only Chlamy & Humans, only Chlamy & Arabidopsis, and all of Chlamy & Humans & Arabidopsis?

A
  • only homologs to arabidopsis: likely related to photosynthesis, etc.
  • only homologs to humans: likely related to flagella, etc.
  • homologs to both arabidopsis and humans: likely related to general eukaryotic function?
18
Q

Basic organization and functional features of the eyespot.

A
  • Enables Chlamy to gain information from its surroundings
  • Layers of carotenoid granule layers under chloroplast membrane and cell plasma membrane which has channelrhodopsin complexes on it
  • Carotenoid layer acts as a reflector so that any light that passed through the plasma membrane can be reflected onto the channelrhodopsin (increasing #protons captured)
19
Q

Structure & function of channelrhodopsin and voltage gated Na channel.

A

Channelrhodopsin is a light-gated channel anchored to the eyespot. When closed, Ca2+ ion cannot enter cell (impermeable), but when channelrhodopsin absorbs a photon, Ca2+ ions can move into the cell and depolarizes the cell. Membrane repolarized by a voltage-gated Na+ channel.

20
Q

Basics of how an action potential is generated (cell is polarized…triggers depolarization then repolarization).

A

Action potential generated when channelrhodopsin absorbs a photon causing its Ca2+ gate opens, allowing calcium ions to flow into the cell and depolarize it, which causes the voltage-gated Na+ channel to open, allowing sodium ions to leave the cell and repolarize it, returning it to its OG polarization (negative charge). This action potential is propagated along the length of the cell.

21
Q

Channelrhodopsin is a photoreceptor….pigment (retinal) and protein (opsin)..

A

Channelrhodopsin is a photoreceptor - all photoreceptors have a protein and a pigment component. In channelrhodopsin, the pigment is retinal, while the protein is opsin.

22
Q

Structural characteristics of pigment molecules.

A

Pigment molecules (e.g. chlorophyll) have a conjugated system of alternating double and single bonds, that allow electrons able to interact with light.

23
Q

Define light absorption and fundamental principles of light absorption by a pigment (in this case we look at chlorophyll). Chl. vs Chl*

A

Light absorption refers to the process where an electron absorbs a proton and ascends to a more excited state (increased energy, indicated by an asterisk).

Each photon excites only one electron.

24
Q

What is an absorption spectrum….chlorophyll or retinal as examples…what does it tell you?

A

Chlorophyll can absord a RED photon and reach a lower excited state OR a BLUE photon and reach a higher excited state, but decays to a lower excited state due to heat loss.
Chlorophyll absorbs strongly in the blue and red wavelengths and very weakly in the green wavelengths BC chlorophyll electrons can only absorb a photon if the increase in energy state matches the exact photon energy.
Retinal has a single excitation peak in the blue parts of the light spectrum.

25
Q

You should know the relative energies of blue, green and red photons of light.

A

Blue ~ 450-495nm
Green ~ 495-570nm
Red ~620-750nm

26
Q

Fundamentals of photoisomerization of retinal and its link to a change in opsin shape (conformation)

A

Retinal absorbs light and changes form from trans retinal to cis retinal. A kink is created at the end of the isomer, where one of the electrons in a double bond gets excited and becomes a single bond, swiveling that part of the molecule, before the electron decays back to the low energy state and the double bond reforms in a new position.

The photon causes a change in shape of the pigment (retinal), which forces a conformational change (shape) of the protein (opsin), which causes the gate in the protein to open.

27
Q

A rod or cone cell is a non-motile cilium! Understand the location of photoreceptor molecules (rhodopsin) in these cells.

A

Photoreceptor molecules form disk that stack up in cilium of rod/cone cells.

28
Q

Key similarities and differences between structure and function of channelrhodopsin in the eyespot and rhodopsin in the human eye.

A
  • Both channelrhodopsin and rhodopsin are created with the retinal pigment and the opsin protein.
    (Light causes cis-retinal to change to trans-retinal in rhodopsin, but trans to cis in channelrhodopsin.) - Can Tigers Really Twerk? Crazy Cats!
  • Functionally the same, both change shape with photon absorption!
  • However, rhodopsin is not an ion channel!! Instead, when it changes shape, it activates a G protein complex which signals the voltage-gated Na+ channels in the plasma membrane to open - more complicated than channelrhodopsin, more proteins involved!
  • Rhodopsin: action potential also runs down plasma membrane of rod/cone cell just like for channelrhodopsin and Chlamy plasma membranes
29
Q

Rhodopsin and channelrhodopsin are analogous structure….look at evolutionary relationship of type 1 vs type 2 opsins.

A

Channelrhodopsin, with a type 1 opsin, is evolutionarily related to bacteria rhodopsin, while eye rhodopsin, with a type 2 opsin, is one of 2 major classes of G-protein coupled receptors.
Both of them independently recruited retinal as the pigment component as they evolved.

30
Q

Distinction between photochemistry as it occurs in eyes and eyespots compared to how it occurs within a photosystem in photosynthesis.

A

Photochemistry is a change in chemical structure caused by the absorption of light.
Two types of photochemistry:
1) changing isomers of substance for photoreceptors
2) oxidation of substance (like chlorophyll), losing an electron for the functioning of a photosystem

31
Q

Optogenetics…what are we trying to do by expressing chlamy opsin in brain cells ?

A

By expressing channelrhodospin (also chlamy opsin) in brain neurons and stimulating them with blue light (causing them to fire action potentials), we can map brain activity.

32
Q

Optogenetic experiment….understand the basics steps involved

A

1) Promoter driving expression is attached to gene encoding for chlamy opsin protein
2) Construct is inserted into virus, which acts as a stable method of transport
3) Virus injected into animal brain and the opsin is expressed in targeted neurons
4) Optrode (fibre-optic cable plus electrode) inserted into brain
5) Thin beam of blue light (laser) opens ion channel in neurons, triggering action potential in specific part of the brain
6) Then, electrophysiological and behavioural results can be recorded and analyzed

33
Q

PHOTOTAXIS…can you think of a single mutation to a structure of process that would cause cells to lose there phototactic ability…as well as Why would cells move away from light.

A
  • Phototaxis: movement towards (+’ve phototaxis) or away (-ive phototaxis) from a light source.
  • Gene encoding for dynein may be mutated, resulting in inability to move
  • Chlamy may move away from the light if it is too strong - causes damage to PSII (specifically D1)