“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