er presentation-updated
TRANSCRIPT
“Direct observation of molecular arrays in the organized smooth
endoplasmic reticulum”
Vladimir M. KorkhovBenoît Zuber
Jason DillonDecember 9, 2010
Bio 441
Background and Terms• ER
– Rough– Smooth
• Evolutionary conservation– Yeast to mammals– 50-100 nm luminal
intermembrane distance
• Tubes vs. Sheets– Cell cycles
Matthew Damstrom Organelles Project. Web. <http://liquidbio.pbworks.com/w/page/11135266/Matthew-Damstrom-Organelles-Project>.
Organized Smooth ER (OSER)• Cubic• Tubular• Stacked sheets• Membrane & organelle biogenesis• Comprises part of nuclear envelope• Peripheral ER – microtubules and
membrane sheets
Possible Stabilizers
• Reticulons and DP1 (tubule stabilization)–Induce high membrane curvature
• Nuclear envelope–Flat double sheet–SUN prot.
• Span NE lumen (nucleus to cytoskeleton via Nesprin)
More Possibilities• Weak interactions, fluorescent prot. tags
– On ER-resident prot.’s (cytoch.-b5, Sec61)– May stabilize ER sheets– May induce OSER formation
• Peripheral ER sheets– Climp63
• Microtubule-binding protein (binds to cytoskeleton)
Calnexin
• Assists protein conformation/folding• Lectin chaperone with a single
transmembrane-spanning domain• Overexpression
–Induces stacked OSER membranes• Very dynamic
OSER Membrane Expansion
• Emery-Dreiffuss disease• Torsion dystonia• Hodgkin’s lymphona• Response to stress
–Excessive malformed proteins
Intent of Experiment
• Identify mol.’s inducing organization of smooth ER sheets
• OSER membrane stracks highly regular• Ordered tethering of membranes
–Large native complex (unknown)–Not through heterologously
overexpressed proteins
Materials
• DMEM and standard cell-culture reagents• SUN1 and SUN2 antibodies• Nesprin-1 antibody• Anti-rabbit antibody conjugated with Texas
Red• Climp63-GFP construct• Calnexin constructs
Methods• HEK293 cells
– Cultured at 37°C– Supplemented with 5% CO2
• Transfections – Lipofectamine-2000– 24 μg of plasmid DNA
• Individual transfection reaction per 3 cm plate– 10 μg of plasmid DNA
• Transfect cells growing in 10 cm dishes
Confocal Microscopy• Poly-l-glutamate-coated glass cover-slips
–Grown until 5080% confluence• Transfection/fixation
–Cells fixed w/ 4% paraformaldehye• PO4-buffered saline (PBS)
–Permeabilized 30 min.’s @ room temp.• PBS w/ 1% BSA & 0.01% Triton-X100
–Stained 1 hr. with 1o & 2o antibodies
Confocal Microscopy contd.
• Stained coverslips washed 4x in PBS–Mounted on glass slides in Vectashield
medium• Images acquired by Zeiss LSM 510
confocal microscope–63x obj. lens
Cryo-electron Microscopy of Vitreous Sections (CEMOVIS)
• Cells centrifuged 5 min.’s @ 1400 rpm– Resuspended in 30% dextran-PBS
• Cells introduced into 200μm deep cavity of copper membrane carrier– Vitrified by high pressure freezing
• Membrane carriers clamped into specimen holder– Trimmed in pyramidal shape w/ diamond knife
CEMOVIS• Cryosections collected on 1000-mesh
grids–Coated w/C–Stored in liquid N
• Tecnai T12 microscope–Film–2,600,030,000x
Intermembrane Distances
• Images scanned and digitized• OSER membranes stacked parallel & perp.• Intermembrane distances measured
–~30% compression due to cutting–Fourier transformation/filtered image
calculation• Cutting by diamond knife did not affect
selected regions
Results• GFP-tagged memb. proteins (hypothesized)
– Weakly interact w/ each other – may lead to stacking• Intrinsic extramembranous domains of ER
proteins• Self-association
• Fluorescent prot. dimerization not a pre-requisite– Calnexin-mCherry fusion as potent as
YFP-/CFP-calnexin
Co-expression of Calnexin- and Climp63-GFP (HEK293)
• Climp63– Contains extended luminal coiled-coil domain– Large, rod-shaped aggregates– Possible stacking of OSER membranes
• No colocalization• Climp63-GFP
– Not found in calnexin-GFP positive multilamellar bodies
– Doesn’t stabilize OSER stacks
LINC Complex & Nesprin 1• Nuclear Envelope proteins
– SUN1 & SUN2 connect inner NE with outer– Cells over-expressing calnexin– Endogenous SUN proteins excluded from calnexin-
induced OSER memb.’s• Endogenous Nesprin-1
– NE to actin cytoskeleton– Spectrin repeats --> possible oligomerization– Excluded from calnexin-CFP stained OSER memb.
Figure 1 – Confocal Microscopy
•OSER membrane biogenesis sustained by monomericfluorescent protein fusion expression •Doesnot involve Climp63, SUN, or Nesprin1 proteins
CEMOVIS Analysis of HEK293 Cells
• Over-expressing calnexin-YFP• 55 vitreous section micrographs• Intermembrane spacing uniformity
– Cytosolic & luminal compartments– 25.3 nm b/t cytosolic, 36.5 nm (perpendicular)– 38.6 nm b/t luminal, 49.8 nm (perpendicular)
Stabilization
• Over-expressed proteins (i.e. calnexin) not enough
• Luminal domain length of calnexin too short
Figure 2 - Luminal and Cytosolic Distances
Black arrowheads demonstrate cytosolic space
Open arrow = cutting direction
CEMOVIS Micrographs
• Proteins packed tighter in OSER than peripheral ER
• Closely spaced arrays of globular complexes at cytosolic face– On outermost membrane– Trapped inside– Complexes still unknown (less than ½ size of
ribosome)
Figure 3 – Molecular Arrays at OSER membranes
F. Dynamic, high flexibility
20nm
G. Plot of angle vs. intermembrane distance
Conclusions• CEMOVIS imaging technique preserved in vivo-
like conditions (hydrated)• Ordered cytosolic and luminal macromolecular
arrays (complexes)– OSER stacking
• Fluorescent protein dimerization does not lead to induction of OSER sheets
• ER-localized proteins may act in stabilization– Cytochrome B5, HMG-CoA reductase
New Proposed Model
• OSER membranes are stabilized by extended arrays of “adhesion” molecules– Less ordered than desmosome junctions
• Identity remains unknown• Still unknown whether “adhesion” molecules
induce OSER stack formation, or stabilize after formation