Cellular Evolution Of Microbial Eyes Flashcards
Eyes have evolved
All over the TOL!
How do eyes work?
- light occlusion/refractory layer
- light sensing surface (rhodopsin proteins)
- juxtaposition of 2 layers provides light detection and direction perception
- light refracts off occlusion layer and hits light sensing surface
- signals generate an action potential
Eye core function
- modified and elaborated on
- vast diversity
eye spots
Eye like systems also all over the TOL
Warnowiid microbial eyes
- dinoflagellates, related to Symbiodinium
- HUGE genome; visible under light microscopy; wrapped around liquid crystals
- oscilloid
Oscilloid
Eye-like structure
- thought it was a squid eye that had become detached
- unculturable
Oscilloid organelles
- TEM
- retinal body
- lens structure
- cornea (crystal)
Retinal bodies
- isolation and micro dissection
- mRNA amplification
- high transcriptional enrichment
Oscilloid hypothesis
- chloroplast derived?
FIBSEM
- focussed ion beam SEM
- takes 100s of hours
1. high pressure freeze
2. SEM
3. Laser a layer off
4. Repeat 1.2.3
5. Colour every cell section; builds a 3D structure
FIBSEM obs?
- retinal body resembles plastid matrix, shaped around lens structure
- ordered structures; compound eyes (rods and cones analog)
- molecular convergence of organised photoreceptor
Proteome reconstruction
- missing PSII; non-photosynthetic
- retained some PS apparatus
- generates proton/ion gradients across membranes
- proton pumps generate proton gradients through ETCs (may not support ATP synthesis); cyclic EF
- drives action potential (light driven proton pump); similar to rhodopsins
How can Warnowiid do without photosynthesis?
- ecology!
- under time-lapse
- cell shoots harpoons/pistons under activation
- snares a bacterium for consumption
Fungi?
Subcellular compartment: fungal zoospore
Blastocladiomycota
- form phototactic zoospores
Blastocladiella
- RGC gene fusion
- localised to SBC (visualised under Nile Red lipid stain)
- pharmacological inhibition of R/GC domain inhibits phototaxis
RGC
- Rhodopsin guanylyl Cyclase
- externalised along lipid droplet surface; forms rhodopsin layer
- lipids refract the light
SBC
- side body complex
- lipid filled organelle
Animal light perception pathway
- Light triggers animal type II rhodopsin
- G protein transducer complex stimulates cGMP hydrolysis
- Na+ influx blocked via closure
- Membrane hyper polarisation
- Synaptic signalling
Fungal light perception pathway
- Light triggers bacteria-type I rhodopsin
- Activates GC
- cGMP synthesis
- K+ channel opened
- Membrane hyperpolarisation
- Action potential activates flagellum beating
Chytrids
- possess a wide range of functionally diversified RGC paralogs
- homo and heterodimers
- gene duplications
- can detect wide range of wavelengths
- optogenetics!
Chloroplasts
W red colourisation ; uses rhodopsin layer
Opsin
- can modify highly cell- spatial- or temporal- specific neutral networks
- microbial derived
- can be used in optogenetics
- introduced after a promoter region by a viral carrier
Optogenetics
- Different light wavelengths are used to initiate neuronal activation/inhibition or receptor-mediated intercellular signalling
- a useful biotechnology and neurobiological tool
How does optogenetics work?
Illumination is delivered to the skull cortex by light delivery to an optic fibre, stabilised by an optic canyon and cranioplastic cement
Blue light
- Triggers ChR2 to rapidly depolarise a neurone
- generates an action potential throughout mono and Divalent cation permeability
ChR2
Channel rhodopsin 2
Optogenetics basics
- type I bacterial rhodopsin and GC domain fusion protein are expressed in animal cells
- induces cGMP into an action potential
- tuneable to a range of wavelengths
GC domain fusion protein
Cyl Op / RGC
Chytrid fungi optogenetics network
Highly distributed
Under comparative analysis of zoospore cellular structures, reconstructing evolution shows
An adaptive radiation of various microtubular forms
How do eye-like systems translate into movement?
Tracking fungal zoospores:
1. Hundreds of Rhizoclasmatium globosum
2. One Chytriomyces hyalinus
Swimming tracks
- B. emersonii (Blastocladiomycota): random walking
- S. punetatus (Chytridiomycota); random walking
- R. globosum (Chytridiomycota); circular pattern
Analysing swimming tracks
- reorientation angle distribution / mean^2 displacement
- shorter and longer timescale motility dynamics vary across groups
A relationship between cytoskeleton and swimming tracks?
Confocal image
Confocal imaging
- 4x Blastocladiomycota, 8x Chytridiomycota
- random walkers: prominent tubulin cytoplasmic structures
- circular swimmers: N/A
- nocodazole creates circular swimmers
Nocodazole
- microtubule pharmacologic
- M: cytoskeleton ablation, diffuse cell, eye organelle collapse
Cytoskeleton is
Necessary
B. emersonii
- random walker
- resembles a light-activated, subcellular “muscle”
- phototactic
- uniformly turns towards eye organelle
B. emersonii EM:
- shows cell system diversification across evolution
- Chytridiomyces comferrae: MLC
- B. emersonii: SBC
MLC
microbody-lipid globule complex
Ultrastructure expansion microscopy (UExM)
1) parasite sedimentation (+/- fixation)
2) cross linking prevention: anchoring
3) gelation
4) denaturation
5) 1st expansion round
6) gel shrinkage, immunostaining
7) final expansion round, imaging
UExM observations
- Confocal
- B. emersonii - random walking (tubulin and centrin)
- Rhizoclosmatium globosum: circular swimming
- microtubules join flagellum
- lipid escape during expansion
Spatial proteomics
1) cell fractionation
2) organelle fractionation
3) peptide generation and TMT labelling
4) high pH reversed phase UPLC
5) data analysis (protein distributions, pRoloc, supervised machine learning)
tSNE plots
- Be dLOPIT data; separate organelles emerge: rhodopsins next to each other; validation
