L1: Subcellular turnover and microscopy

Degradation of cell components: Two mainpathways of protein-containing degradation

1. Ubiquitin-proteosome system

   • for single unfoldable proteins

2. Autophagy

   • less digestible proteins

   • organelles

   • macromolecule complexes

1. Ubituitin-proteasome system

For single unfolded polypeptides

1. Recognised as misfolded

2. tagged by covalent additios of poly-ubiquitin side chains

3. recognised by proteasome (barrel-shaped multiprotein complex)

4. fed through for degradation

What is the protasome also used to degrade?

ER-associated degradation (ERAD)

1. ER lumen proteins are proteolysed into peptides

2. translocated retrogradely into cytosol

3. for proteasomal degradation

2. Autophagy and its role

• ‘self eating’

• Lysosomes degrade cellular components→ form autophagosomes i the the lysosome

• Are induced by amino-acid starvation

Its role:

• suggesting a role is responsing to amino acid storages

How discovered/ investigated

• ‘Autophagic' bodes’ accumulates in yeast vacuoles with imparied protease activity

• Loss of ABs can be used to screen for mutants with defective autophagy

• Recovery of ATG mutants (apg in yeast) or blockage using drugs identified

  • components of autophagy machinery

  • and defined steps depending of where autophagy was blocked

What are the steps in Autophagy

note: can use GFP to visualise the AB

Note: Macroautophagy vs Microautophagy vs Chaperone-mediated

1. Macro→ normal ‘autophagy’

2. Micro→ invagination of autophagic substrates into autophagosomes

   • doubled up by lysosomes

3. Chaperone mediated→ translocation of chaperone-bound denatured substrates from the cytosol across the lysosome membrane into the lysosome

   • i.e the protein is translocated in directly

Autophagy can be divided into two types depending on what its substrates are

1. Constitutive

   • many cell components can be degraded

   • non specific

   • but, overall level is controlled by need (e.g need to recycle nutrients)

2. Substrate-specific

   • targeted

   • specific organelles are recognised by autophagy machinery

     • autophagy adaptors that recognise ‘eat-me’ signals on surface of substrate

   • when there is a physiolgical need to remove that specific organelle/substrate

e.g mitochondria autophagy→ mitophagy

2. Types of ‘eat-me’ signals

1. Proteins covalently modified by covalent attachent of ubiquitin protein

2. decrepit organelles

3. toxic protein aggregates (found in many neurodegenertaive disease

4. intracellular pathogens

Roles of autophagy

1. Starvation

2. Aggregates (protein and RNA)

3. Organelles: mitophagy, ERphagy, lipophagy

What is autophagy controlled by

Must be appropriate substrates and conditions

Controlled by:

• mTORC1

• autophagy adaptors

How was the regulation of autophagy investigated and findings

Rapamycin:

• Found to be inducer of autophagy

• Investigated finding its cellular targets:

  • rapamysin-resistant mutants of yeast

Eventual findings:

• Cellular target contained a large protein kinase, Tor

  • Tor→ ‘target of rapamysin’

  • mTor→ mammalian Tor

What is mTor and its role

• Protein kinase

• forms two alternative complexes:

  1. mTORC1

  2. mTORC2

These two have different effects but overall key in signalling

1. Sensing availability of energy and nutrients

2. Regulating cellular responses to nutrient availability

How does tapamycin work on this?

1. Rapamysin binds to FKPB12 (another subunit of mTORC1 complex)

2. makes mTor activity in mTORC1 sensitive to acute rapaysin exposure

Effects of mTORC1 vs mTORC2

1. mTORC1→ Promotes growth (blocks autophagy)

2. mTORC2→ enables autophagy?

Complementary roles

Nutrient regulation of mTORC1

There are multiple pathways in regulating activity in response to energy or aa availability:

1. Nutrients→ RagA/C (GTPases)→ lysosomal translocation→ mTORC1→ growth

2. Insulin→ Rheb→ kinase activation→ mTORC1→ growth

Amino acid detection

• Converge on rag

• different sensors for different aa

Ultimately sensing nutrients on the surface of the lysosome

Regulation of autophagy by mTORC1

In general:

1. mTORC1 phsphylates some early players in the autophagy pathway

2. This is important in regulating constituitive autophagy

When high nutrients:

1. high mTORC1 actvity

2. Inhibits autophagy

3. promotes cell growth and high biosynthetic activity

In famine:

1. low mTORC1

2. disinhibits autophagy

3. breaking down cell components

4. provide new sources of aa, nucleotides, lipids for essential cell function in absence of external sources

Evidence for mTORC1 role in autophagy: diseases caused by mutation to mTORC1

1. e.g Drosophila Tor mutants

2. Human TSC1/TSC2 mutations in tuberous sclerosis

   • number of growths/cysts in many organs

   • from excess mTORC1 activity

3. Neurodegneration (ALS, FTD) by various autophagy mutants

   • defective clearance of protein/RNA aggregates

But how is mTorc1 itself regulated? Localisation

Localisation→ seen with immunofluorescent miscopy

1. In starvation→ mTORC1 is localised to the cytosol inactive

2. When aa available→ activate→goes to surface of lysosomes

3. Here is it regulated by all signallig machinery on the lysosome surface that respond to signals of

   • aa abundance (Via Rag/Regulator pathway)

   • glucose

   • oxygen

   • growth factors (via TSC/Rheb pathway)

4. Lysosomes themselves are dynamic

   • transported by MT based motors

   • aa availability can lead to relocalization from peri-nuclear to the cell periphery

Lysosomes as an amino acid store

• degerneative activity makes them rich stores of amino acids

• must be tightly regulated by

  • nutritional state

  • local needs in the cell

Example of lysosome store regulation and evidence

1. Release aa to cytoplasm

2. activate mTORC1

EVIDENCE

• mutating a channel that releases aa from lysosome to the cytosol

• blocks mTORC1 activation

Conclusion:

• one way TORC1 is regulatede if through lysosome aa efflux to signal nutrient availability

How can mTORC1 regulate the lysosome

In starvation

1. mTORC1 inhibition

2. reduces efflux of aa from lysosome

3. converts lysosome into a cellular store of essential aa for essential protein synthesis

Overall roles of lysosomes

1. Protein degradation (aminio acid source)

2. Amino acid store

   • linking store with cell nutritional status→ mTORC1 signalling

   • highly localised store for used for protein synthesis

3. A Ca2+ store

   • can release Ca2+ on demand

   • via interactions with lysosomes (see next lecture)

Other nutritional signalling pathways besides mTor

1. AMPK

   1. ATP demand rise

   2. ATP fall

   3. AMP rises

   4. activates AMPK (AMP-dependent protein kinase)

   5. phosphorylates mutliple targets to restore mitochondrial function/biosynthesis

   6. reduce ATP consumption

2. Insulin (physlogenetically ancient signallig pathway)

   • adapts metablsm and physiology

   • at cellular and organismal level

   • to nutritial states of high carb/lipid

Seeing inside organelles: types of fluoresecent miscrocopy methods

1. Widefield

2. Confocal

1. Widefield

• whole smaple illuminated

• out-of-focus light doesn’t form an image

  • but still reaches detector

2. Laser scanning confocal microscopy

overall: light through a pinhole to cut out unfocused light

• single point illuminated with bright laser

• rapid scanning of laser across the sample builds up an image

• Pinhole in the detector light path

  • allows all light from focus plane through

• Out-of-focus light mostly excluded by pinhole→ image is an optical section

• Smaller pinhole→ thinner optical section (but less bright)

In order to image sub-organelle resolution we need and why

Super-resolution microscopy:

• most organelles at 1um to few um

• slightly larger than the theoretical limit of resolution of light microscopy

  • (half the wavelength of visible light (diffraction limit))

• So organelles can just about be seen with light but

  • sub-organelle is hard to distinguish

Point spread function and what is it dependent on

• limit of resolution

• forms a spread-out image

  • with the point source being the centre of the PSF

What dependent on:

1. refractive index

2. wavelength

3. numerical aperture of objective

4. optical properties

Two types of Super-resolution microscopy

1. Single-molecule localisation microscopy (SMLM)

2. Stimulated depletion Emission Microscopy (STED)

1. What is the basis of SMLM

1. Activate one molecule at a time

   • With photo-activatable or photo-switchable dyes/proteins

   • ativated randomly at low efficiency by short pulse of a specific wavelength

   • produces a single molecule pixel

2. Record where PSF of this single molecule is

3. Photoswitch it off by a different wavelength

4. Process repeated (and a different pixel will be activated)

5. The middle of the pixel must be where the actual image is

6. repeat many times to get idea of all of the pixels i nthe image

1. resolution of this method

• resolve a few 10s of nm

1. Limitations of this method

• number of rounds of photoswitching

• hard to apply repeats to live imaging

• takes time

2. STED: stimulated depletion Emission Microscopy: concept

1. Use a doughnut-shaped ring of high-intsensity far-red light

2. this suppresses fluorescene of the outer parts

3. increases the resolution

2. How does this red ring work to for this?

• Normal fluoresecence:

  • return of an electron to S0 ground state gives normal fluorescence

• With red ring

  • smaller energy loss for falling electrons

  • longer-waventlgeth is outputted

  • which is not detected (not visible light)

2. Pros and cons

Pros:

• does not need repeated round

• resolution→ 50nm

• increasing power of doughnut can decrease this further

Cons/limits and solution

• limit to how far power can increase without bleaching fluorphores

  • solution: use chemically modified proteins whose fluorescence survives better than GFP

• Uses a lot of energy and heat??

Other types of super-resolution approaches

1. Structured Illumintion Microscopy

   • gentler on live preparations than STED

   • faster than SMLM

   • comutationally complex

   • depth limited to few um

How is works:

• contrast raw images (below the diffraction limit) from Moire patterns *above the diffraction limit)

• Take single image in different oritentations to get more and more info of what the original pattern was with computational analysis

Results: Conventional vs super-resolution images

Instead of super-resoluton: Expansion microscopy

If you cannot bring resolution to prep, bring the prep to the resolution

• impregnate preparation with a gel that expands on hydration

• with light protease treatment

• to allow uniform expansion of cell components

More microscoptic methods for viewing organelles

1. Light sheet

2. Lattice light-sheet

why are these methods used instead of confocal sometimes?

• decreases the photodamage from confocal

  • by using a single plane of light

  • in contrast→ confocal illuminates the whole plane

1. Light sheet

• sample if illuminated by sheet of light perpendicular to the imaging objective

  • (rather than via)

• good alternative to confocal microscopy for imaging single planes

  • SPIM→ Single-plane illumination microscopy

1. Advantages of SPIM

1. reduces photodamage by confining illumination to the focal place

2. much faster than confocal

   • since laser only has to scan in one axis

   • x (not x and y)

2. Lattice light sheet

• uses multiple beams (lattice)

• to spread a thin light-sheet out across the entire imaging place

• since it is not continuous in the x-axis→ it must be dithered to make a continuous sheet

2. Pros

• further gains in speed and phototoxicity

How can multiple organelles be live imaged: why can’t just use 6 different markers for 6 organelles?

• Requires 6 different fluorescent protein tags

however:

• with so many tags→ emission spectra cannot be separated using silter sets alone

Solution to this: Spectral imaging

1. Dispersion grating separates emission light into wavelentghs across the entire spectrum

2. parallel detectors ample emission across the entire spectrum

3. Code of colour sorresponds to different organelles from 6 fulorophores

How to decode this code for the organelles (Spectral unmixing)

1. Each channel detects mix of signals from 6 fluorophores

2. here 26 simulatenous equations to find the 6 unknowns