ER Sheets Research Articles

The CCT chaperonin and actin modulate the ER and RNA-binding protein condensation during oogenesis and maintain translational repression of maternal mRNA and oocyte quality.

The regulation of maternal mRNAs is essential for proper oogenesis, the production of viable gametes, and to avoid birth defects and infertility. Many oogenic RNA-binding proteins have been identified with roles in mRNA metabolism, some of which localize to dynamic ribonucleoprotein granules and others that appear dispersed. Here, we use a combination of in vitro condensation assays and the in vivo Caenorhabditis elegans oogenesis model to characterize the properties of the conserved KH-domain MEX-3 protein and to identify novel regulators of MEX-3 and three other translational regulators. We demonstrate that MEX-3 undergoes phase separation and appears to have intrinsic gel-like properties in vitro. We also identify novel roles for the chaperonin-containing tailless complex polypeptide 1 (CCT) chaperonin and actin in preventing ectopic RNA-binding protein condensates in maturing oocytes that appear to be independent of MEX-3 folding. The CCT chaperonin and actin also oppose the expansion of endoplasmic reticulum sheets that may promote ectopic condensation of RNA-binding proteins. These novel regulators of condensation are also required for the translational repression of maternal mRNA which is essential for oocyte quality and fertility. The identification of this regulatory network may also have implications for understanding the role of hMex3 phase transitions in cancer.

Roles of Tubulin Concentration during Prometaphase and Ran-GTP during Anaphase of Caenorhabditis elegans Meiosis.

In many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in Caenorhabditis elegans oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF, but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes.

Roles of Tubulin Concentration during Prometaphase and Ran-GTP during Anaphase of C. elegans meiosis.

In many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in C. elegans oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes.

INPP5K and Atlastin-1 maintain the nonuniform distribution of ER-plasma membrane contacts in neurons.

In neurons, the ER extends throughout all cellular processes, forming multiple contacts with the plasma membrane (PM) to fine-tune neuronal physiology. However, the mechanisms that regulate the distribution of neuronal ER-PM contacts are not known. Here, we used the Caenorhabditis elegans DA9 motor neuron as our model system and found that neuronal ER-PM contacts are enriched in soma and dendrite and mostly absent in axons. Using forward genetic screen, we identified that the inositol 5-phosphatase, CIL-1 (human INPP5K), and the dynamin-like GTPase, ATLN-1 (human Atlastin-1), help to maintain the non-uniform, somatodendritic enrichment of neuronal ER-PM contacts. Mechanistically, CIL-1 acts upstream of ATLN-1 to maintain the balance between ER tubules and sheets. In mutants of CIL-1 or ATLN-1, ER sheets expand and invade into the axon. This is accompanied by the ectopic formation of axonal ER-PM contacts and defects in axon regeneration following laser-induced axotomy. As INPP5K and Atlastin-1 have been linked to neurological disorders, the unique distribution of neuronal ER-PM contacts maintained by these proteins may support neuronal resilience during the onset and progression of these diseases.

Calumenin-1 Interacts with Climp63 to Cooperatively Determine the Luminal Width and Distribution of Endoplasmic Reticulum Sheets.

SummaryThe ER is composed of distinct structures like tubules, matrices, and sheets, all of which are important for its various functions. However, how these distinct ER structures, especially the perinuclear ER sheets, are formed remains unclear. We report here that the ER membrane protein Climp63 and the ER luminal protein calumenin-1 (Calu1) collaboratively maintain ER sheet morphology. We show that the luminal length of Climp63 is positively correlated with the luminal width of ER sheets. Moreover, the lumen-only mutant of Climp63 dominant-negatively narrows the lumen of ER sheets, demonstrating that Climp63 acts as an ER luminal bridge. We also reveal that Calu1 specifically interacts with Climp63 and antagonizes Climp63 in terms of both ER sheet distribution and luminal width. Together, our data provide insight into how the structure of ER sheets is maintained and regulated.

Mechanosensitive Fluorescent Probes to Image Membrane Tension in Mitochondria, Endoplasmic Reticulum, and Lysosomes.

Measuring forces inside cells is particularly challenging. With the development of quantitative microscopy, fluorophores which allow the measurement of forces became highly desirable. We have previously introduced a mechanosensitive flipper probe, which responds to the change of plasma membrane tension by changing its fluorescence lifetime and thus allows tension imaging by FLIM. Herein, we describe the design, synthesis, and evaluation of flipper probes that selectively label intracellular organelles, i.e., lysosomes, mitochondria, and the endoplasmic reticulum. The probes respond uniformly to osmotic shocks applied extracellularly, thus confirming sensitivity toward changes in membrane tension. At rest, different lifetimes found for different organelles relate to known differences in membrane organization rather than membrane tension and allow colabeling in the same cells. At the organelle scale, lifetime heterogeneity provides unprecedented insights on ER tubules and sheets, and nuclear membranes. Examples on endosomal trafficking or increase of tension at mitochondrial constriction sites outline the potential of intracellularly targeted fluorescent tension probes to address essential questions that were previously beyond reach.

Uncovering Theorganelle Interactome: Dynamic Imaging of Multiple Organelles

Numerous aspects of cell biology and biophysics are becoming clearer due to emerging visualization technologies, which can capture processes at the level of whole organisms down to single molecules. Here, I will discuss developments in probes and microscopes that are dramatically expanding productive imaging. To surmount fluorophore bleed-through, a combined excitation-based spectral unmixing and lattice light sheet microscopy was used to visualize up to six organelles (i.e., ER, Golgi, mitochondria, lysosomes, peroxisomes and lipid droplets) simultaneously within cells. This allowed us to track these organelles through time and analyze their inter-organelle contacts, providing a systems-level map of the organelle interactome and how it is perturbed under different physiological conditions. To increase temporal resolution during imaging, we employed total internal reflection fluorescence combined with structured illumination microscopy to visualize organelle dynamics at very high temporal-spatial resolution. Examining the ER, we observed that many peripheral ER sheets seen using diffraction-limited imaging are actually highly perforated structures comprised of tightly latticed groups of dynamic tubules. Within the latticed ER tubule meshwork, subdiffraction-limited holes were observed (∼150-250 nm diameter) having transient lifespans (∼250 msec). Viewed at higher resolution using lattice light sheet microscopy combined with point accumulation for nanoscale topology (PAINT), the peripheral ER sheets represented a complex meshwork of tightly cross-linked ER tubules, with potentially important roles in ER function.

Postmitotic nuclear pore assembly proceeds by radial dilation of small membrane openings.

The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and the structures of assembling NPCs in a correlative 3D EM time course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets whose holes progressively shrink. NPC precursors are found in small membrane holes and dilate radially during assembly of the inner ring complex, forming thousands of transport channels within minutes. This mechanism is fundamentally different from that of interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport competent.

A family of membrane-shaping proteins at ER subdomains regulates pre-peroxisomal vesicle biogenesis

Saccharomyces cerevisiae contains three conserved reticulon and reticulon-like proteins that help maintain ER structure by stabilizing high membrane curvature in ER tubules and the edges of ER sheets. A mutant lacking all three proteins has dramatically altered ER morphology. We found that ER shape is restored in this mutant when Pex30p or its homologue Pex31p is overexpressed. Pex30p can tubulate membranes both in cells and when reconstituted into proteoliposomes, indicating that Pex30p is a novel ER-shaping protein. In contrast to the reticulons, Pex30p is low abundance, and we found that it localizes to subdomains in the ER. We show that these ER subdomains are the sites where most preperoxisomal vesicles (PPVs) are generated. In addition, overproduction or deletion of Pex30p or Pex31p alters the size, shape, and number of PPVs. Our findings suggest that Pex30p and Pex31p help shape and generate regions of the ER where PPV biogenesis occurs.

Probing Short-Range Protein Brownian Motion in the Cytoplasm of Living Cells

The translational motion of small molecules in cells appears to be suppressed compared to what is observed in dilute solutions. Although, the rotation of small proteins is almost unhindered, pointing out a local aqueous environment. Different theoretical models provide explanations for this apparent discrepancy but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive experimental test of the nature of the translational motion in cells is still missing owing to the lack of techniques capable of probing protein motion with the required temporal and spatial resolution. We show that fluorescence-fluctuation analysis of raster scans at variable time scales can provide this information. By using GFP, we measure protein translational motion at the unprecedented time-scale of 1 microsecond, unveiling unobstructed Brownian motion from 25 to 100 nanometers, and partially-suppressed diffusion above 100 nm. Experiments on in vitro model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. In this regard, internal membranes (e.g. the ER sheets, vesicles, Golgi apparatus, etc.) appear to be the more likely candidates as selective disruption of the microtubules network by treatment with Nocodazole did not significantly alter GFP behavior in the cytoplasm. Also, the same measurement in a structurally-different (e.g devoid of membranes) intracellular environment, such as the nucleoplasm, yields a different behavior, in which GFP motion is never coincident with that in a dilute solution. Finally, we believe the present findings coupled with use of genetically-encoded fluorescent markers pave the way to novel studies of biomolecular processes in live cells at the physiologically-relevant spatio-temporal scale. Supported by grants NIH P41-GM103540 and NIH P50-GM076516 (grants to EG), MIUR under FIRB-RBAP11X42L and Fondazione Monte dei Paschi di Siena (grants to FB).

Fluorescence Fluctuation Microscopy Techniques to Study mRNA Synthesis and Dynamics

1627-Pos Board B578 Probing Short-Range Protein Brownian Motion in the Cytoplasm of Living Cells Carmine Di Rienzo 1,2 , Enrico Gratton 3 , Fabio Beltram 1,2 , Francesco Cardarelli 2 . NEST, Scuola Normale Superiore, Pisa, Italy, 2 Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Pisa, Italy, 3 Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering., University of California, Irvine, CA, USA. The translational motion of small molecules in cells appears to be suppressed compared to what is observed in dilute solutions. Although, the rotation of small proteins is almost unhindered, pointing out a local aqueous environment. Different theoretical models provide explanations for this apparent discrepancy but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive experimental test of the nature of the translational motion in cells is still missing owing to the lack of techniques capable of probing protein motion with the required temporal and spatial resolution. We show that fluorescence-fluctuation analysis of raster scans at variable time scales can provide this information. By using GFP, we measure protein translational motion at the unprecedented time-scale of 1 micro- second, unveiling unobstructed Brownian motion from 25 to 100 nanometers, and partially-suppressed diffusion above 100 nm. Experiments on in vitro model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. In this regard, internal membranes (e.g. the ER sheets, vesicles, Golgi apparatus, etc.) appear to be the more likely candidates as selective disruption of the microtubules network by treatment with Nocodazole did not significantly alter GFP behavior in the cytoplasm. Also, the same measurement in a structurally-different (e.g devoid of membranes) intra- cellular environment, such as the nucleoplasm, yields a different behavior, in which GFP motion is never coincident with that in a dilute solution. Finally, we believe the present findings coupled with use of genetically-encoded fluores- cent markers pave the way to novel studies of biomolecular processes in live cells at the physiologically-relevant spatio-temporal scale. Supported by grants NIH P41-GM103540 and NIH P50-GM076516 (grants to EG), MIUR under FIRB- RBAP11X42L and Fondazione Monte dei Paschi di Siena (grants to FB).

STIM1L traps and gates Orai1 channels without remodeling the cortical ER.

STIM proteins populate and expand cortical endoplasmic reticulum (ER) sheets to mediate store-operated Ca2+ entry (SOCE) by trapping and gating Orai channels in ER-plasma membrane clusters. A longer splice variant, STIM1L, forms permanent ER-plasma membrane clusters and mediates rapid Ca2+ influx in muscle. Here, we used electron microscopy, total internal reflection fluorescence (TIRF) microscopy and Ca2+ imaging to establish the trafficking and signaling properties of the two STIM1 isoforms in Stim1−/−/Stim2−/− fibroblasts. Unlike STIM1, STIM1L was poorly recruited into ER-plasma membrane clusters and did not mediate store-dependent expansion of cortical ER cisternae. Removal of the STIM1 lysine-rich tail prevented store-dependent cluster enlargement, whereas inhibition of cytosolic Ca2+ elevations or removal of the STIM1L actin-binding domain had no impact on cluster expansion. Finally, STIM1L restored robust but not accelerated SOCE and clustered with Orai1 channels more slowly than STIM1 following store depletion. These results indicate that STIM1L does not mediate rapid SOCE but can trap and gate Orai1 channels efficiently without remodeling cortical ER cisternae. The ability of STIM proteins to induce cortical ER formation is dispensable for SOCE and requires the lysine-rich tail of STIM1 involved in binding to phosphoinositides.

Reticulon 4 Is Necessary for Endoplasmic Reticulum Tubulation, STIM1-Orai1 Coupling, and Store-operated Calcium Entry

Despite recent advances in understanding store-operated calcium entry (SOCE) regulation, the fundamental question of how ER morphology affects this process remains unanswered. Here we show that the loss of RTN4, is sufficient to alter ER morphology and severely compromise SOCE. Mechanistically, we show this to be the result of defective STIM1-Orai1 coupling because of loss of ER tubulation and redistribution of STIM1 to ER sheets. As a functional consequence, RTN4-depleted cells fail to sustain elevated cytoplasmic Ca(2+) levels via SOCE and therefor are less susceptible to Ca(2+) overload induced apoptosis. Thus, for the first time, our results show a direct correlation between ER morphology and SOCE and highlight the importance of RTN4 in cellular Ca(2+) homeostasis.

Microstructure and mechanical properties of Al–Mg–Er sheets jointed by friction stir welding

1.7mm thick cold-rolled Al–Mg–Er alloy sheets under as-rolled and annealed conditions were subjected to friction stir welding (FSW). The as-rolled sheet showed fibrous tissues with a high density of dislocations. After annealing, the fibrous tissues were replaced by coarse, nearly-equiaxed grains ∼18μm in size and the density of dislocations decreased greatly, resulting in significantly reduced hardness of the annealed sheet. After FSW, the nugget zone (NZ) of both the FSW joints of as-rolled and as-annealed sheets consisted of fine equiaxed recrystallised grains with sizes of 4.5μm and 4.3μm, respectively. For the joint of the as-rolled sheet, the dislocations in the NZ and thermomechanically affected zone (TMAZ) were significantly reduced and the NZ showed lower hardness than the base material (BM). However, for the joint of the as-annealed sheet, the NZ exhibited higher hardness than the BM due to significant grain refinement in the NZ. The joint of the as-rolled sheet showed a joint efficiency of 68% and fractured in the NZ but the joint of the as-annealed sheet had a joint efficiency of nearly 100% and fractured in the BM.

Untangling the web: Mechanisms underlying ER network formation

The ER is a continuous membrane system consisting of the nuclear envelope, flat sheets often studded with ribosomes, and a polygonal network of highly-curved tubules extending throughout the cell. Although protein and lipid biosynthesis, protein modification, vesicular transport, Ca2+dynamics, and protein quality control have been investigated in great detail, mechanisms that generate the distinctive architecture of the ER have been uncovered only recently. Several protein families including the reticulons and REEPs/DP1/Yop1p harbor hydrophobic hairpin domains that shape high-curvature ER tubules and mediate intramembrane protein interactions. Members of the atlastin/RHD3/Sey1p family of dynamin-related GTPases interact with the ER-shaping proteins and mediate the formation of three-way junctions responsible for the polygonal structure of the tubular ER network, with Lunapark proteins acting antagonistically. Additional classes of tubular ER proteins including some REEPs and the M1 spastin ATPase interact with the microtubule cytoskeleton. Flat ER sheets possess a different complement of proteins such as p180, CLIMP-63 and kinectin implicated in shaping, cisternal stacking and cytoskeletal interactions. The ER is also in constant motion, and numerous signaling pathways as well as interactions among cytoskeletal elements, the plasma membrane, and organelles cooperate to position and shape the ER dynamically. Finally, many proteins involved in shaping the ER network are mutated in the most common forms of hereditary spastic paraplegia, indicating a particular importance for proper ER morphology and distribution in large, highly-polarized cells such as neurons. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.

Role for cER and Mmr1p in Anchorage of Mitochondria at Sites of Polarized Surface Growth in Budding Yeast

Mitochondria accumulate at neuronal and immunological synapses and yeast bud tips and associate with the ER during phospholipid biosynthesis, calcium homeostasis, and mitochondrial fission. Here we show that mitochondria are associated with cortical ER (cER) sheets underlying the plasma membrane in the bud tip and confirm that a deletion in YPT11, which inhibits cER accumulation in the bud tip, also inhibits bud tip anchorage of mitochondria. Time-lapse imaging reveals that mitochondria are anchored at specific sites in the bud tip. Mmr1p, a member of the DSL1 family of tethering proteins, localizes to punctate structures on opposing surfaces of mitochondria and cER sheets underlying the bud tip and is recovered with isolated mitochondria and ER. Deletion of MMR1 impairs bud tip anchorage of mitochondria without affecting mitochondrial velocity or cER distribution. Deletion of the phosphatase PTC1 results in increased Mmr1p phosphorylation, mislocalization of Mmr1p, defects in association of Mmr1p with mitochondria and ER, and defects in bud tip anchorage of mitochondria. These findings indicate that Mmr1p contributes to mitochondrial inheritance as a mediator of anchorage of mitochondria to cER sheets in the yeast bud tip and that Ptc1p regulates Mmr1p phosphorylation, localization, and function.

Quasimonochromatic x‐ray computed tomography by the balanced filter method using a conventional x‐ray source

A quasimonochromatic x-ray computed tomography (CT) system utilizing balanced filters has recently been developed for acquiring quantitative CT images. This system consisted of basic components such as a conventional x-ray generator for radiography, a stage for mounting and rotating objects, and an x-ray line sensor camera. Metallic sheets of Er and Yb were used as the balanced filters for obtaining quasimonochromatic incident x rays that include the characteristic lines of the W Kalpha doublet from a tungsten target. The mean energy and energy width of the quasimonochromatic x rays were determined to be 59.0 and 1.9 keV, respectively, from x-ray spectroscopic measurements using a high-purity Ge detector. The usefulness of the present x-ray CT system was demonstrated by obtaining spatial distributions of the linear attenuation coefficients of three selected samples--a 20 cm diameter cylindrical water phantom, a 3.5 cm diameter aluminum rod, and a human head phantom. The results clearly indicate that this apparatus is surprisingly effective for estimating the distribution of the linear attenuation coefficients without any correction of the beam-hardening effect. Thus, implementing the balanced filter method on an x-ray CT scanner has promise in producing highly quantitative CT images.

Effect of Two Furanocoumarins and Three Other Coumarins on Ultrastructure, ATPases and Acid Phosphatases in Meristematic Cells of Allium cepa Root Tips

The structure of the cytoplasm in root tip meristematic cells of Allium cepa was affected by coumarin, 4-hydroxycoumarin, 7-hydroxycoumarin, psoralen, and xanthotoxin, leading to segregation of areas of cytoplasm by sheets of ER and invaginations of plasmalemma. Such areas either contained more ribosomes or were autophagic. Mitochondria divided after treatment and enlarged to the size of mega-mitochondria. Their matrix was mostly electron dense and cristae were numerous and dilated, suggesting changes in respiration processes. Furanocoumarins affected ATPases and acid phosphatases differently from coumarin and hydroxycoumarins. Furanocoumarins increased the activity of ATPases compared with the control, whereas other coumarins decreased this activity. Acid phosphatases showed increased activity after treatment with coumarin and hydroxycoumarins, but furanocoumarins had no significant effect on these enzymes.

Immunodetection of cytoskeletal structures and the Eg5 motor protein on deep‐etch replicas of Xenopus egg cortices isolated during the cortical rotation

We have developed a new method for immunogold detection on deep-etch replicas of isolated Xenopus egg cortices in order to examine the interactions of different cortical elements in three dimensions at high resolution. We have applied this technique to vegetal cortices isolated during the second half of the first cell cycle. The vegetal cortical region at this time is the site of cellular machinery responsible for the 'cortical rotation'. The entire cortex translocates with respect to the inner cytoplasm, relocating dorsalising determinants to the future dorsal side of the egg. The aligned microtubules in the shear zone between cytoplasm and cortex, implicated in the cortical rotation, were found to be organised as interweaving loose bundles. Interleaved amongst these aligned microtubules were extensive sheets of ER lying in layers parallel to the egg surface. Cytokeratin filaments were found to associate closely with the microtubules over short stretches. Putative actin filaments were present in the shear zone and in the cortex. Eg5, an abundant kinesin-related microtubule motor protein, and candidate for a role in generating cortical rotation movement, showed an almost exclusive localisation to microtubules. Immunofluorescence studies of cortices treated with detergent to disrupt ER or cold to depolymerise microtubules confirmed that Eg5 associates primarily with microtubules. We propose revised models for the mechanism of cortical rotation based on these observations and conclude that Eg5 is unlikely to move ER relative to microtubules during the cortical rotation.

Immunodetection of cytoskeletal structures and the Eg5 motor protein on deep-etch replicas of Xenopus egg cortices isolated during the cortical rotation

We have developed a new method for immunogold detection on deep-etch replicas of isolated Xenopus egg cortices in order to examine the interactions of different cortical elements in three dimensions at high resolution. We have applied this technique to vegetal cortices isolated during the second half of the first cell cycle. The vegetal cortical region at this time is the site of cellular machinery responsible for the ‘cortical rotation’. The entire cortex translocates with respect to the inner cytoplasm, relocating dorsalising determinants to the future dorsal side of the egg. The aligned microtubules in the shear zone between cytoplasm and cortex, implicated in the cortical rotation, were found to be organised as interweaving loose bundles. Interleaved amongst these aligned microtubules were extensive sheets of ER lying in layers parallel to the egg surface. Cytokeratin filaments were found to associate closely with the microtubules over short stretches. Putative actin filaments were present in the shear zone and in the cortex. Eg5, an abundant kinesin-related microtubule motor protein, and candidate for a role in generating cortical rotation movement, showed an almost exclusive localisation to microtubules. Immunofluorescence studies of cortices treated with detergent to disrupt ER or cold to depolymerise microtubules confirmed that Eg5 associates primarily with microtubules. We propose revised models for the mechanism of cortical rotation based on these observations and conclude that Eg5 is unlikely to move ER relative to microtubules during the cortical rotation.