This document summarizes research into the structure of organized smooth endoplasmic reticulum (OSER) membranes. Key findings include: 1) Cryo-electron microscopy of vitreous sections revealed ordered cytosolic and luminal protein arrays between closely spaced OSER membranes, suggesting these stabilize the membrane stacks. 2) Overexpression of proteins like calnexin induced OSER sheet formation, but fluorescent tags on these proteins were not required, implying other ER-resident proteins may be involved in stabilization. 3) The identity of stabilizing "adhesion molecules" forming the ordered arrays between membranes remains unknown.

1. “Direct observation of molecular
arrays in the organized smooth
endoplasmic reticulum”
Vladimir M. Korkhov
Benoît Zuber
Jason Dillon
December 9, 2010
Bio 441

2. 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>.

3. Organized Smooth ER (OSER)
• Cubic
• Tubular
• Stacked sheets
• Membrane & organelle biogenesis
• Comprises part of nuclear envelope
• Peripheral ER – microtubules and
membrane sheets

4. 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)

5. 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)

6. Calnexin
• Assists protein conformation/folding
• Lectin chaperone with a single
transmembrane-spanning domain
• Overexpression
–Induces stacked OSER membranes
•Very dynamic

7. OSER Membrane Expansion
• Emery-Dreiffuss disease
• Torsion dystonia
• Hodgkin’s lymphona
• Response to stress
–Excessive malformed proteins

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. CEMOVIS
• Cryosections collected on 1000-mesh
grids
–Coated w/C
–Stored in liquid N
• Tecnai T12 microscope
–Film
–2,600,030,000x

15. 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

16. 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

17. 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

18. 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.

19. Figure 1 – Confocal Microscopy
•OSER membrane
biogenesis sustained
by monomeric
fluorescent protein
fusion expression
•Does
not involve Climp63,
SUN, or Nesprin1
proteins

20. 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)

21. Stabilization
• Over-expressed proteins (i.e. calnexin) not
enough
• Luminal domain length of calnexin too short

22. Figure 2 - Luminal and Cytosolic
Distances
Black arrowheads
demonstrate cytosolic
space
Open arrow =
cutting direction

23. 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)

24. Figure 3 – Molecular Arrays at
OSER membranes

25. F. Dynamic,
high flexibility
20nm
G. Plot of angle vs.
intermembrane distance

26. 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

27. 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