Distinctive subcellular alterations induced by hypertonic stress in sea urchin eggs

Sea urchin eggs continuously exposed to a hypertonic solution were ultrastructurally examined for osmotic-stress induced alterations. No fertilization membranes formed during the treatment and the surface-cortex complexes remained unaltered from the unfertilized state. However, the osmotic stress did induce a number of subcellular changes. During the first 30 minutes of the treatment the eggs formed many endoplasmic reticulum whorls and compacted Golgi body aggregations. Both of these new formations can be correlated with rapid changes in intracellular calcium, known to occur in hypertonic stressed eggs. Aggregations of mitochondria could be observed at later stages; these aggregations can also be related to subcellular stress and possible changes in internal calcium concentrations. The various morphological transitions within the cytoplasm, along with the lack of a cortical reaction in these eggs, not only supports the idea that calcium is released during parthenogenetic activation, but also suggests that this free calcium originates from stores other than the stores that are involved during fertilization or simple artificial activation.     

Proper symmetric and asymmetric endoplasmic reticulum partitioning requires astral microtubules

Mechanisms that regulate partitioning of the endoplasmic reticulum (ER) during cell division are largely unknown. Previous studies have mostly addressed ER partitioning in cultured cells, which may not recapitulate physiological processes that are critical in developing, intact tissues. We have addressed this by analysing ER partitioning in asymmetrically dividing stem cells, in which precise segregation of cellular components is essential for proper development and tissue architecture. We show that in Drosophila neural stem cells, called neuroblasts, the ER asymmetrically partitioned to centrosomes early in mitosis. This correlated closely with the asymmetric nucleation of astral microtubules (MTs) by centrosomes, suggesting that astral MT association may be required for ER partitioning by centrosomes. Consistent with this, the ER also associated with astral MTs in meiotic Drosophila spermatocytes and during syncytial embryonic divisions. Disruption of centrosomes in each of these cell types led to improper ER partitioning, demonstrating the critical role for centrosomes and associated astral MTs in this process. Importantly, we show that the ER also associated with astral MTs in cultured human cells, suggesting that this centrosome/astral MT-based partitioning mechanism is conserved across animal species.     

COPII mitigates ER stress by promoting formation of ER whorls

Cells mitigate ER stress through the unfolded protein response (UPR). Here, we report formation of ER whorls as an effector mechanism of the ER stress response. We found that strong ER stress induces formation of ER whorls, which contain ER-resident proteins such as the Sec61 complex and PKR-like ER kinase (PERK). ER whorl formation is dependent on PERK kinase activity and is mediated by COPII machinery, which facilitates ER membrane budding to form tubular-vesicular ER whorl precursors. ER whorl precursors then go through Sec22b-mediated fusion to form ER whorls. We further show that ER whorls contribute to ER stress-induced translational inhibition by possibly modulating PERK activity and by sequestering translocons in a ribosome-free environment. We propose that formation of ER whorls reflects a new type of ER stress response that controls inhibition of protein translation.Subject terms: Endoplasmic reticulum, Collective cell migration     

Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg

During the first cell cycle of the ascidian egg, two phases of ooplasmic segregation create distinct cytoplasmic domains that are crucial for later development. We recently defined a domain enriched in ER in the vegetal region of Phallusia mammillata eggs. To explore the possible physiological and developmental function of this ER domain, we here investigate its organization and fate by labeling the ER network in vivo with DiIC16(3), and observing its distribution before and after fertilization in the living egg. In unfertilized eggs, the ER-rich vegetal cortex is overlaid by the ER-poor but mitochondria-rich subcortical myoplasm. Fertilization results in striking rearrangements of the ER network. First, ER accumulates at the vegetal-contraction pole as a thick layer between the plasma membrane and the myoplasm. This accompanies the relocation of the myoplasm toward that region during the first phase of ooplasmic segregation. In other parts of the cytoplasm, ER becomes progressively redistributed into ER-rich and ER- poor microdomains. As the sperm aster grows, ER accumulates in its centrosomal area and along its astral rays. During the second phase of ooplasmic segregation, which takes place once meiosis is completed, the concentrated ER domain at the vegetal-contraction pole moves with the sperm aster and the bulk of the myoplasm toward the future posterior side of the embryo. These results show that after fertilization, ER first accumulates in the vegetal area from which repetitive calcium waves are known to originate (Speksnijder, J. E. 1992. Dev. Biol. 153:259-271). This ER domain subsequently colocalizes with the myoplasm to the presumptive primary muscle cell region.     

ESCRTing endoplasmic reticulum to microautophagic degradation

ABSTRACT

Changing conditions necessitate cellular adaptation, which frequently entails adjustment of organelle size and shape. The endoplasmic reticulum (ER) is an organelle of exceptional morphological plasticity. In budding yeast, ER stress triggers the de novo formation of ER subdomains called ER whorls. These whorls are selectively degraded by a poorly defined type of microautophagy. We recently showed that ESCRT proteins are essential for microautophagic uptake of ER whorls into lysosomes, likely by mediating the final scission of the lysosomal membrane. Furthermore, ER-selective microautophagy acts in parallel with ER-selective macroautophagy. The molecular machineries for these two types of autophagy are distinct and their contributions to ER turnover vary according to conditions, suggesting that they serve different functions. Our study provides evidence for a direct role of ESCRTs in microautophagy and extends our understanding of how autophagy promotes organelle homeostasis.     

Structural reorganizations of the endoplasmic reticulum during egg maturation and fertilization

The endoplasmic reticulum (ER) of eggs is a major internal store of calcium ions that must be properly mobilized at fertilization for development to proceed. In most species, the ER develops distinct clusters in the cortical ooplasm as the oocyte matures into a fertilizable egg. Following fertilization, the structure of the ER rapidly reorganizes in eggs that produce a single fertilization-induced calcium wave, whereas ER clusters persist for relatively long periods in eggs that generate multiple calcium oscillations. This review considers such pre- and post-fertilization reorganizations of the ER and what effects these changes might have on calcium signaling patterns.     

A Cytochemical Study of the Sulfhydryl Groups of Sea Urchin Eggs during the First Cleavage

In the eggs of four species of echinoderms, Mespilia globulus, Pseudocentrotus depressus, Hemicentrotus pulcherrimus and Clypeaster japonicus, changes in the distribution of protein-bound SH groups from fertilization to the 2 cell stage have been studied cytochemically by use of a mercaptide-forming azo dye. In the eggs of these species, the color intensity in the cytoplasm increased upon fertilization. The astral centers and spindle during mitosis were stained deeply. When the aster formation was suppressed by ether, hyaline spots appeared in the egg cytoplasm instead of well formed astral centers and these spots were stained by the SH-specific dye. Upon recovery of such eggs in pure sea water, and when cleavage ensued, such spots disappeared and two new astral centers were reorganized. The SH-protein occurring in the centrosphere is considered to be the precursor material for the asters and spindle, and this material is apparently derived from the cytoplasm.     

TMTC1 and TMTC2 Are Novel Endoplasmic Reticulum Tetratricopeptide Repeat-containing Adapter Proteins Involved in Calcium Homeostasis

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.     

Cyclin B-cdk1 controls pronuclear union in interphase

In sexual reproduction, the union of the male and female pronuclei occurs in fertilized eggs to mix genetic materials derived from both parents, thereby creating a new genome for the next generation [1-4]. The process leading to pronuclear union consists of pronuclear congression, which depends on astral microtubules derived from sperm centrosome [5-8], and the subsequent pronuclear fusion or karyogamy. The union process progresses in parallel with the first embryonic cell cycle, but the molecular mechanisms involved are poorly understood. Here, we devise a labeling method with Dendra2 to track both pronuclei individually in living starfish eggs. Although pronuclear union naturally proceeds while G1 arrest is released by fertilization and S phase progresses [9], we show that the cell-cycle resumption and progression are not prerequisites for pronuclear union. However, low levels of cyclin B- (but not cyclin A-) Cdk1 activity are detectable even in interphase, and are indispensable for pronuclear union, by contributing at least to pronuclear congression through formation of sperm aster. Pronuclear congression thus requires the activity of M-phase cell-cycle regulator in interphase, independently of the cell-cycle regulation. These findings not only provide a clue to the regulatory aspect of creation of new genome with fertilization, but also reveal a novel role for the M-phase Cdk1 during interphase.     

Calcium ultrastructural localization in Xenopus laevis eggs following activation by pricking or by calcium ionophore A 23187

In the egg of Xenopus laevis a cortical network of smooth endoplasmic reticulum (SER) surrounds and interconnects each cortical granule (CG) (Campanella and Andreuccetti, '77). This network is a possible intracellular site of calcium storage to be called into action for CG exocytosis. In our experiments, Xenopus eggs, unfertilized or activated by pricking or by calcium ionophore A 23187, have been fixed in osmium-pyroantimonate for calcium localization. Our data show that deposits can be detected only in activated eggs. The calcium chelator edetate (EGTA) and x-ray microprobe analysis demonstrate that they contain calcium. Deposits are found on liposomes and on all intraovular cytomembranes, which therefore appear to be possible sites of calcium sequestration. In the case of ionophore-activated eggs, deposits are detectable independently of the presence of extracellular calcium. These data show that in Xenopus at activation an intracellular liberation of calcium occurs similar to that described in other species. Furthermore, the fact that antimony deposits are observed only after activation makes Xenopus eggs appropriate material in which to follow the temporal and spatial sequence of appearance of the deposits during the early stages of activation. Our results show that antimony deposits appear first in SER vesicles between the plasma membrane and CGs and then spread to the rest of the egg cytomembranes. These data corroborate our hypothesis that in Xenopus the cortical SER network is the first intracellular site where calcium is released at activation. The possible mechanism of calcium release and propagation along the egg cortex is discussed.     

The endoplasmic reticulum and calcium storage

Calcium storage is one of the functions commonly attributed to the endoplasmic reticulum (ER) in nonmuscle cells. Several recent studies have added support to this concept. Analysis of reticuloplasm, the luminal ER content, has shown that it contains several proteins (reticuloplasmins) which are prospective calcium storage proteins. One of these, calreticulin, is also present in the sarcoplasmic reticulum (SR). In sea urchin eggs, a calsequestrin-like protein has been clearly localised to the ER. The recent demonstration that the IP3 receptor, which has similarities with the calcium release channel in the SR is also localised in the ER membrane suggests that calcium stored in the ER is important for intracellular signalling. The alternative view, that the physiologically important calcium store is a specialised organelle, the calciosome, is not supported by these observations. Recent evidence also suggests that ER calcium might be important in ER structure and in the retention of the luminal ER proteins.     

Cyclic structural changes in endoplasmic reticulum and Golgi apparatus in the hippocampal neurons of ground squirrels during hibernation

Repetitive remodeling and renewal of the cytoplasmic structures realizing synthesis of proteins accompanies the cycling of ground squirrels between torpor and arousal states during hibernation season. Earlier we have shown partial loss of ribosomes and nucleolus inactivation in CA3 hippocampal pyramidal neurons in each bout of torpor with rapid and full recovery after warming up. Here we describe reversible structural changes in endoplasmic reticulum (ER) and Golgi complex (G) in these neurons. Transformation of ER from mainly cysternal to tubular form and from mainly granular to smooth type occurs at every entrance in torpor, while the opposite change occurs at arousal. Torpor state is also associated with G fragmentation and loss of its flattened cisternae. Appearance in torpor of the autophagosomal vacuoles containing fragments of membrane structures and ribosomes is a sign of their partial destruction. Granular ER restoration, perhaps through assembly from the multilamellar membrane structures, whorls or bags, begins as early as in the middle of the torpor bout, while G flattened cisternae reappear only at warming. ER and G completely restore their structure 2-3 hours after the provoked arousal. Thus, hibernation represents and example of nerve cell structural adaptation to alterations in functional and metabolic activity through both active destruction and renewal of ribosomes, ER, and G. Perhaps, it is the incomplete ER autophagosomal degradation at torpor provides its rapid renewal at arousal by reassembly from the preserved fragments.     

RAB-11 permissively regulates spindle alignment by modulating metaphase microtubule dynamics in Caenorhabditis elegans early embryos

Alignment of the mitotic spindle along a preformed axis of polarity is crucial for generating cell diversity in many organisms, yet little is known about the role of the endomembrane system in this process. RAB-11 is a small GTPase enriched in recycling endosomes. When we depleted RAB-11 by RNAi in Caenorhabditis elegans, the spindle of the one-cell embryo failed to align along the axis of polarity in metaphase and underwent violent movements in anaphase. The distance between astral microtubules ends and the anterior cortex was significantly increased in rab-11(RNAi) embryos specifically during metaphase, possibly accounting for the observed spindle alignment defects. Additionally, we found that normal ER morphology requires functional RAB-11, particularly during metaphase. We hypothesize that RAB-11, in conjunction with the ER, acts to regulate cell cycle-specific changes in astral microtubule length to ensure proper spindle alignment in Caenorhabditis elegans early embryos.     

Presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the spindle, the diastema and the cleavage furrow in the blastomere of the Xenopus embryo

The present study examines the indispensability of a nucleus or nucleus-deriving factors in the induction of cleavage in Xenopus eggs by testing cleavage in Xenopus eggs fertilized with ultraviolet (UV)-damaged sperm and deprived of the female nucleus. These eggs, which contain only one UV-damaged nucleus with one set of centrioles, undergo unique cleavages. Cleavage takes place in only one of the two blastomeres formed by the immediately preceding cleavage. Histologically, only one nucleus, which does not appear to be organized into typical chromosomes, is found in one of the two blastomeres formed by the immediately preceding cleavage. The typical bipolar spindle and the diastema, or a slit of astral rays, are formed in the blastomere that contains the nucleus. By contrast, only asters lacking the spindle and the diastema are formed in the remaining blastomeres, which do not contain a nucleus. The same results are obtained in eggs that contain two UV-damaged nuclei with one set of centrioles. In these eggs, cleavage appears to occur in one or two blastomeres that contain either or both of the nuclei and one bipolar spindle. In eggs that contain one intact and one UV-damaged nuclei, cleavage takes place quite normally with each blastomere containing one nucleus or one set of chromosomes as well as one bipolar spindle. Thus, there is a very close correlation between the presence of a nucleus and the formation of the mitotic spindle, the diastema and the cleavage furrow in the blastomeres of Xenopus embryos. We conclude that the presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the bipolar spindle, the diastema and the cleavage furrow in the blastomeres of the Xenopus embryos.     

Cytological changes and DNA and protein synthesis in parthenogenetically activated sea urchin eggs

Summary The present study deals with cytological observations, DNA and protein synthesis in artificially activated sea urchin eggs. The eggs were activated by means of Loeb's double treatment with butyric acid and hypertonic sea water. Most of the eggs ofHemicentrotus pulcherrimus divided when the chromosomes duplicated after formation of the first monaster and other eggs divided at a later cell cycle. In the eggs ofTemnopleurus toreumaticus, however, haploid division at the first cell cycle was observed predominantly.Activated eggs that were treated for 25 min with hypertonic sea water showed a marked uptake of³H-thymidine during the two periods of 30–40 min and 90–100 min after the double treatment. These periodic changes in the³H-thymidine uptake paralleled morphological changes within the nucleus. However, these periods of increased uptake were not observed in the eggs treated with hypertonic sea water for 60 min. During exposure to hypertonic sea water, the³H-thymidine-uptake by eggs activated with butyric acid decreased gradually. When the uptake of¹⁴C-valine by eggs was measured, a very low level was seen in unfertilized eggs. The level of uptake increased strikingly when the eggs were activated with butyric acid but was suppressed by the hypertonic treatment. However, removal of the eggs to sea water allowed the uptake to return to the former high level. This pattern suggests that the hypertonic treatment has an inhibitory effect on the synthesis of protein (or enzymes) which obstruct cleavage induction.     

Calreticulin expression levels and endoplasmic reticulum during late oogenesis and early embryogenesis of <Emphasis Type="Italic">Rhodnius prolixus</Emphasis> Stahl

This study reports the cloning, expression analysis and localization of calreticulin (CRT) in the endoplasmic reticulum (ER) during late oogenesis and early embryogenesis of the insect Rhodnius prolixus. CRT was cloned and sequenced from cDNA extracted from unfertilized eggs. Real-time PCR showed that CRT expression remains at lower levels during late oogenesis when compared to vitellogenic oocytes or day 0 laid fertilized eggs. Immunofluorescence microscopy showed that this protein is located in the periphery of the egg, in a differential peripheral ooplasm surrounding the yolk-rich internal ooplasm, only identified by transmission electron microscopy (TEM) of thin sections. Using immunogold electron microscopy, the ER ultrastructure (CRT labeled) was identified in the peripheral ooplasm as dispersed lamellae, randomly distributed in the peripheral ooplasm. No massive alterations of ER ultrastructure were found before or right after (30 min) fertilization, but an increase in CRT expression levels and assembly of typical rough ER (parallel cisternae with associated ribosomes) were observed 18–24 h after oviposition. The lack of ER assembly at fertilization and the later formation of rough ER together with the increase in CRT expression levels, suggest that the major functions of ER might be of great importance during the early events of development. The possible involvement of ER in the early steps of embryogenesis will be discussed.     

Microinjected Polystyrene Beads Move Along Astral Rays in Sand Dollar Eggs

Movements of polystyrene beads along astral rays of the sperm aster and the mitotic aster were investigated in eggs of the sand dollars, Clypeaster japonicus and Scaphechinus mirabilis . Polystyrene beads injected into the unfertilized egg were at a standstill in the protoplasm. After fertilization, these beads exhibited movements toward the center of the sperm aster along the rays, and finally gathered around the astral center. They were distributed in blastomeres together with the mitotic centers during successive cleavages. When injected into eggs during mitosis, beads moved to the centers of the mitotic asters along astral rays. The injected beads did not move when the aster was disorganized by treatment with Colcemid, and moved when it formed after UV-irradiation. These results indicate that microtubules of astral rays are essential to the movement of polystyrene beads. The movement of small polystyrene beads (0.2–0.3 μm in diameter) resembled the saltatory movement of endogenous cytoplasmic granules, and the movement of large beads (ca. 1 μm in diameter) resembled the female pronuclear migration. All of these movements observed in fertilized eggs were demonstrated to be microtubule-dependent, perhaps sharing the same basic mechanisms.     

The induction of de novo centrioles in sea urchin eggs: a possible common mechanism for centriolar activation among parthenogenetic procedures

Eggs from two species of sea urchins were subjected to a variety of novel two-step parthenogenetic activation procedures. These treatments readily resulted in the formation of cytasters and centrioles. Centrioles are restricted to the cytasters, as well as to the broad perinuclear zone, and it appears that all cytasters contain one or more centrioles. The mode of centriolar origin and maturation, as revealed by these new procedures, is identical to that of centrioles induced by other procedures. Both centriolar and cytastral development are retarded by the parthenogenetic treatments; also, mitosis is generally delayed. These results, along with those of others, reveal that parthenogenetic agents have opposing effects on eggs. The agents are stimulatory in that they activate latent centrioles and the cell cycle, in general. They are inhibitory in that they suppress a variety of physiochemical events. Of these events, only one appears to be common to all parthenogenetic agents: the inhibition of protein synthesis. Egg centrioles may be kept in a latent state by repressor proteins. If these proteins normally cycle rapidly, then an inhibition of their continuous synthesis will disrupt their normal replacement rate. As such, a delay in replacement will allow functioning repressor proteins, associated with latent centrioles, to become nonfunctional, which in turn allows latent centrioles to become derepressed. This deblocking reaction would then activate the appearance of de novo centrioles in eggs.     

Distribution of Membrane-Associated Calcium in Fertilized Sea Urchin Eggs during Mitosis*

The distribution of membrane-associated calcium in dividing sea urchin eggs was examined with chlortetracycline as a fluorescent chelate probe. The fluorescence of bound chlortetracycline in fertilized eggs was initially evenly distributed, but began to gather around the nucleus in prophase, and formed a dumb-bell shaped condensation enclosing the mitotic apparatus by metaphase. During anaphase and telophase, the fluorescence was observed in kinetochore-to-pole regions of the spindle, with little fluorescence in the interkinetochore region. The astral regions showed intense fluorescence. The distribution of the chlortetracycline-fluorescence coincided with that of ER-like membranes seen in electron micrographs. The distribution of the fluorescence was obscure and the birefringence of spindles disappeared on perfusion on perfusion of the cells in metaphase with 1 mM tetracaine, which is known to displace membrane-bound calcium. These results suggest that intracellular free calcium ions are sequestered in the membrane system associated with the mitotic apparatus during mitosis.