You will be reading primary research papers throughout the semester. You are expected to evaluate each paper and turn in a one page review that addresses the following issues: 1) what isthe main point or purpose of this paper? 2) what research question or hypothesis is being tested? 1You will be reading primary research papers throughout the semester. You are expected to evaluate each paper and turn in a one page review that addresses the following issues: 1) what isthe main point or purpose of this paper? 2) what research question or hypothesis is being tested? 13) Identify and describe a pivotal experiment from the paper and what was learned in that specific experiment. 4) identify a weakness in the paper and design a control or series of control experiments that could improve that part of the paper; and 5) if this was your research, what experiment (with controls) would you do next?3) Identify and describe a pivotal experiment from the paper and what was learned in that specific experiment. 4) identify a weakness in the paper and design a control or series of control experiments that could improve that part of the paper; and 5) if this was your research, what experiment (with controls) would you do next?
Organization ofthe Sea Urchin Egg Endoplasmic Reticulum and Its Reorganization at Fertilization Mark Terasaki*§ and LaurindaA. Jaffe*§ * Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892 ; $ DepartmentofPhysiology, University ofConnecticut Health Center, Farmington, Connecticut 06032 ; and §Marine Biological Laboratory, Woods Hole, Massachusetts 02543 Abstract . The ER of eggs of the sea urchin Lytechi- nus pictus was stained by microinjecting a saturated solution of the fluorescent dicarbocyanine DiICtg(3) (DiI) in soybean oil ; the dye spread from the oil drop into ER membranes throughout the egg but not into other organelles . Confocal microscopy revealed large cisternae extending throughout the interior of the egg and a tubular membrane network at the cortex . Since diffusion of DiI is confined to continuous bilayers, the ing most marked by 2-3 min . By 5-8 min the ER re- turned to an organization similar to that of the unferti- lized egg . The cortical network also changed at fertili- zation ; it became disrupted and eventually recovered . DiI labeling allowed continuous observations of the ER during pronuclear migration and mitosis . DiI-stained membranes accumulated in the region of the microtu- bule array surrounding the sperm nucleus and centriole (the sperm aster) as it migrated to the center of the spread of the dye supports the concept that the ER is egg; this accumulation persisted near the centrosomes a cell-wide, interconnected compartment . In time lapse and zygote nucleus throughout pronuclear fusion and observations, the internal cisternae were seen to be in continuous motion, while the cortical ER was station- ary. After fertilization, the internal ER appeared to become more finely divided, beginning as a wave ap- parently coincident with the calcium wave and becom- HE ER is the site of protein synthesis (Palade and Siekevitz, 1956) and lipid synthesis (Wilgram and Kennedy, 1963) and it is one of the intracellular com partments involved in calcium regulation (Streb et al ., 1984 ; Ross et al ., 1989) . The ER has distinct functional domains, such as the nuclear envelope (Watson, 1955), and rough and smooth ER (Palade, 1955) ; there are other morphologically identifiable domains that may have functional significance, such as cisternae and tubules (Palade and Porter, 1954), and cortical and noncortical ER (Porter, 1961) . ER membranes participate in membrane traffic with the Golgi apparatus (Palade, 1975), and have structural interactions with micro- tubules (Terasaki et al ., 1986) and actin filaments (Kachar and Reese, 1988) . In view of all of these characteristics, the ER may be comparable to the plasma membrane in com- plexity of function and perhaps exceeds it in morphological complexity. New information about the distribution ofthe ER has been obtained with the fluorescent dicarbocyanine dye DiOCb(3) (Terasaki et al ., 1984) . This dye permeates the plasma mem- Address correspondence to Mark Terasaki, Building 36, Room 2A-29, NIH, 9000 Rockville Pike, Bethesda, MD 20892 . On request, a videotape of the results described in this paper will be provided at cost . © The Rockefeller University Press, 0021-9525/91/09/929/12 $2 .00 The Joumal of Cell Biology, Volume 114, Number 5, September 1991929-940 929 the first two mitotic cycles . We have used a new meth- od to observe the spatial and temporal organization of the ER in a living cell, and we have demonstrated a striking reorganization of the ER at fertilization . brane and stains many if not all intracellular membranes ; it has been primarily useful in the periphery of cultured cells where the ER is a single two dimensional layer that can be easily distinguished from other membranes (Terasaki, 1989) . To investigate the three dimensional distribution of ER in living cells, we have adapted a method that has been used for visualizing the complex form of neurons in intact tissues . This method for staining the plasma membrane in- volves introducing a longer alkyl chain fluorescent dicar- bocyanine dye "DiI" (DUC,e(3) or DUC43)) into contact with the cells ; the lipophilic dye transfers into the plasma membrane and diffuses within the membrane bilayer through- out the neuronal processes (Honig and Hume, 1986, 1989 ; see also Haugland, 1989) . This dye has recently been used to stain the ER and sarcoplasmic reticulum in broken cell preparations where DiI aggregates contact and incorporate in the membrane bilayer and then diffuse within it (Henson et al ., 1989 ; Baumann et al ., 1990 ; Terasaki, M., J. H . Henson, D. A . Begg, B . Kaminer, and C. Sardet, unpub- lished results) . To apply a source of DiI to the membrane of the ER in an intact living cell, we dissolved the dye in soy- bean oil (Wesson cooking oil) and microinjected the dye- saturated oil into the cytoplasm of sea urchin eggs . Using confocal microscopy (White et al ., 1987), we found that the on M arch 15, 2018 jcb.rupress.org D ow nloaded from http://jcb.rupress.org/ dye spread through the ER, but not into other organelles, thus allowing us to visualize the structure of the ER in the egg . Previous studies in fixed preparations have provided evi- dence for transformations in ER organization that accom- pany physiological changes in oocytes and eggs. A structural reorganization ofthe ER occurs during oocyte maturation of the frog Xenopus (Gardiner and Grey, 1983 ; Campanella et al ., 1984 ; Charbonneau and Grey, 1984; Larabell and Chandler, 1988) ; the ER cisternae come to surround the cor- tical granules, establishing the ER in a form and location that may serve to release calcium and cause cortical granule exo- cytosis at fertilization . A similar development of tubular ER around the cortical granules occurs between the vitellogenic oocyte and the mature egg in the sea urchin (Henson et al ., 1990) . Indications of changes in ER structure at fertilization include the finding that the ER appears to be disrupted on cortices derived from recently fertilized sea urchin eggs (Sandet, 1984; Henson et al ., 1989) ; a question remains however whether this is a change in the ER or a change in the cortex that causes it to shear differently when the cortex is prepared after fertilization . In Renopus, electron micros- copy has shown that junctions between the cortical ER and plasma membrane decrease at fertilization (Gardiner and Grey, 1983) and in another frog, Discoglossus, fertilization results in a rearrangement ofthe subcortical ER near the ani- mal pole (Campanella et al ., 1988) . Using the DiI-oil drop injectiontechnique, we have now made continuous observa- tions ofthe rearrangements ofthe ER in the living sea urchin egg during fertilization . We find that within the first 20 min after fertilization, the ER throughout the egg cytoplasm un- dergoes a dramatic sequence of structural changes . Materials and Methods Sea urchins (Lytechinus pictus) were obtained from Marinus, Inc. (Venice, CA) ; this species was used because of the optical clarity of its eggs . Eggs and spermwere obtained by injection of0.5 M KCI into the coelomic cavity. The gametes were suspended in artificial sea water. Experiments were per- formed at 22-24°C . DiICIS (3) : 1,1'-dioctadecyl-3,3,3;3'-tetramethylindocarbocyanine per- chlorate (DiI)l was obtained from Molecular Probes (Eugene, OR) . A saturated solution of Dil in oil was made by mixing several crystals of Dil in 100 pl of soybean oil (Wesson oil ; obtained from Food Buoy, Woods Hole, MA, and Sutton Place Gourmet, Bethesda, MD; lots from both sources behaved similarly) . The solution was kept atroom temperature, and was used over a period of several days . The Dil solution was microinjected into eggs held between parallel cov- erslips of number zero thickness (Kiehart, 1982) . The eggs were observed during injection using a standard Zeiss upright microscope with a IOx phase contrast objective . The oil solution was microinjected withaconstric- tion pipette connected to a micrometer syringe (Hiramoto, 1974 ; Kishi- moto, 1986) . After injection, the chamber holding the eggs was transferred to the stage of the confocal microscope. Insemination was accomplished by introducing a sperm suspension through the open side of the injection chamber. Eggs were observed using a laser scanning confocal microscope (Model 600; Bio-Rad Laboratories, Oxnard, CA) with an argon laser for illumina- tion and coupled with a Zeiss Axioplan or a Nikon Optiphot microscope. Observations weremadeusing a Zeiss Planapo 63x N .A . 1.4 objective lens or a Nikon Planapo 60x N.A . 1.4 objective lens. For the observations, the laser was used at full power with a 1 or 3% neutral density filter, and with the confocal aperture between 3 and 5 . Astepper motorwasused for collect- ing Z-series images ; this motor had a minimum step size of 0.18 wm for the Zeiss microscope and 0.1 tm for the Nikon microscope . Images were stored on a Panasonic 3031F optical memory disk recorder 1 . Abbreviation used in this paper: Dil, 1,1'-dioctadecyl-3,3,3',3'-tetra- methylindocarbocyanine perchlorate . The Journal of Cell Biology, Volume 114, 1991 (OMDR) . The OMDR was operated in its on line mode, using macro pro- grams run by the BioRad confocal microscope software . Scale bar calibra- tions were obtained by recording images of a stage micrometer for each zoom setting and scanning speedused, forbothx and y directions. To make the figures, the videomonitor wasphotographed using 35-mmfilm (TMAX 100 ; Eastman Kodak Co., Rochester, NY) . A monitor with an adjustable video image height was used to obtain identical magnification in z and y directions . Results Internal and Cortical ER ofthe Unfertilized Egg After injection into a sea urchin (Lytechinus pictus) egg of Figure 1. Spreading of DiI from an injected oil drop into the ER ofan unfertilized egg. (A) 5 min afterinjection. The dye has moved from the oil drop into the nearby cytoplasm but has not yet spread across the egg . (B) 30 min after injection . The dye has spread throughout the cytoplasm but is still more concentrated near the oil drop . (C) 30 min after injection . An optical section showing the nucleus . Bar, 50 um . 930 on M arch 15, 2018 jcb.rupress.org D ow nloaded from http://jcb.rupress.org/ Figure 2 . ER of the unfertilized egg . An optical section slightly below the top surface of the egg . Cistemae (lamellar sheets) are seen in the central region, with the tubular network at the periphery. Bar, 10 Am . a drop of soybean oil saturated with Dil (4-20 pl, 0.5-3% of the egg volume), the dye spread throughout the egg (110 Am diameter) over a period of ti 30