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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.09.007 Byline: Wassim El-Jouni (a), Shirley Haun (a), Khaled Machaca (a)(b) Keywords: Plasma-membrane Ca.sup.2+ ATPase (PMCA); Calcium; Oocyte maturation; Endocytosis; Trafficking; Lipid-rafts; Xenopus laevis Abstract: A transient increase in intracellular Ca.sup.2+ is the universal signal for egg activation at fertilization. Eggs acquire the ability to mount the specialized fertilization-specific Ca.sup.2+ signal during oocyte maturation. The first Ca.sup.2+ transient following sperm entry in vertebrate eggs has a slow rising phase followed by a sustained plateau. The molecular determinants of the sustained plateau are poorly understood. We have recently shown that a critical determinant of Ca.sup.2+ signaling differentiation during oocyte maturation is internalization of the plasma membrane calcium ATPase (PMCA). PMCA internalization is representative of endocytosis of several integral membrane proteins during oocyte maturation, a requisite process for early embryogenesis. Here we investigate the mechanisms regulating PMCA internalization. To track PMCA trafficking in live cells we cloned a full-length cDNA of Xenopus PMCA1, and show that GFP-tagged PMCA traffics in a similar fashion to endogenous PMCA. Functional data show that MPF activation during oocyte maturation is required for full PMCA internalization. Pharmacological and co-localization studies argue that PMCA is internalized through a lipid raft endocytic pathway. Deletion analysis reveal a requirement for the N-terminal cytoplasmic domain for efficient internalization. Together these studies define the mechanistic requirements for PMCA internalization during oocyte maturation. Author Affiliation: (a) Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA (b) Weill Cornell Medical College in Qatar, Qatar Foundation, Education City, Doha, Qatar Article History: Received 27 June 2008; Revised 14 August 2008; Accepted 8 September 2008

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.10.034 Byline: Wassim El-Jouni, Byungwoo Jang, Shirley Haun, Khaled Machaca Keywords: Oocyte maturation; Xenopus; Ca.sup.2+ signaling; IP.sub.3 receptor; Plasma membrane Ca.sup.2+ ATPase; Endocytosis Abbreviations: Ca.sup.2+.sub.cyt, cytoplasmic Ca.sup.2+; IP.sub.3, d-myo-inositol-1,4,5-trisphosphate; Caged-IP.sub.3, d-myo-inositol 1,4,5-trisphosphate, P4(5)-(1-(2-nitrophenyl)ethyl) ester; IP.sub.3R, IP.sub.3 receptor; GVBD, germinal vesicle breakdown; MPF, maturation promoting factor; SOCE, store operated Ca.sup.2+ entry; ER, endoplasmic reticulum; OG-1, Oregon Green BAPTA-1; PMCA, plasma membrane Ca.sup.2+-ATPase; SERCA, sarco-endoplasmic reticulum Ca.sup.2+-ATPase; EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid; BAPTA, 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; NP-EGTA, o-nitrophenyl EGTA; PIP2, phosphatidylinositol-4,5-bisphosphate Abstract: Ca.sup.2+ is the universal signal for egg activation at fertilization in all sexually reproducing species. The Ca.sup.2+ signal at fertilization is necessary for egg activation and exhibits specialized spatial and temporal dynamics. Eggs acquire the ability to produce the fertilization-specific Ca.sup.2+ signal during oocyte maturation. However, the mechanisms regulating Ca.sup.2+ signaling differentiation during oocyte maturation remain largely unknown. At fertilization, Xenopus eggs produce a cytoplasmic Ca.sup.2+ (Ca.sup.2+.sub.cyt) rise that lasts for several minutes, and is required for egg activation. Here, we show that during oocyte maturation Ca.sup.2+ transport effectors are tightly modulated. The plasma membrane Ca.sup.2+ ATPase (PMCA) is completely internalized during maturation, and is therefore unable to extrude Ca.sup.2+ out of the cell. Furthermore, IP.sub.3-dependent Ca.sup.2+ release is required for the sustained Ca.sup.2+.sub.cyt rise in eggs, showing that Ca.sup.2+ that is pumped into the ER leaks back out through IP.sub.3 receptors. This apparent futile cycle allows eggs to maintain elevated cytoplasmic Ca.sup.2+ despite the limited available Ca.sup.2+ in intracellular stores. Therefore, Ca.sup.2+ signaling differentiates in a highly orchestrated fashion during Xenopus oocyte maturation endowing the egg with the capacity to produce a sustained Ca.sup.2+.sub.cyt transient at fertilization, which defines the egg's competence to activate and initiate embryonic development. Author Affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 West Markham St. Slot 505, Little Rock, AR 72205, USA Article History: Received 29 July 2005; Revised 6 October 2005; Accepted 26 October 2005

Transport effectors of Ca(2+) are tightly modulated during Xenopus oocycte maturation; processes are described for maintaining elevated cytoplasmic Ca(2+) necessary for initiating embryonic development.

The universal signal for egg activation at fertilization is a rise in cytoplasmic [Ca.sup.2+] with defined spatial and temporal kinetics. Mammalian and amphibian eggs acquire the ability to produce such [Ca.sup.2+] signals during a maturation period that precedes fertilization and encompasses resumption of meiosis and progression to metaphase II. In Xenopus, immature oocytes produce fast, saltatory [Ca.sup.2+] waves that can be oscillatory in nature in response to I[P.sub.3]. In contrast, mature eggs produce a single continuous, sweeping [Ca.sup.2+] wave in response to I[P.sub.3] or sperm fusion. The mechanisms mediating the differentiation of [Ca.sup.2+] signaling during oocyte maturation are not well understood. Here, I characterized elementary [Ca.sup.2+] release events ([Ca.sup.2+] puffs) in oocytes and eggs and show that the sensitivity of I[P.sub.3]-dependent [Ca.sup.2+] release is greatly enhanced during oocyte maturation. Furthermore, [Ca.sup.2+] puffs in eggs have a larger spatial fingerprint, yet are short lived compared to oocyte puffs. Most interestingly, [Ca.sup.2+] puffs cluster during oocyte maturation resulting in a continuum of [Ca.sup.2+] release sites over space in eggs. These changes in the spatial distribution of elementary [Ca.sup.2+] release events during oocyte maturation explain the continuous nature and slower speed of the fertilization [Ca.sup.2+] wave. Keywords: Oocyte maturation; Xenopus; [Ca.sup.2+] signaling; IP3-dependent [Ca.sup.2+] release; [Ca.sup.2+] puffs; [Ca.sup.2+] waves: Fertilization; Elementary [Ca.sup.2+] release; [Ca.sup.2+] imaging; Meiosis

Caenorhabditis elegans spermatogenesis is especially suited for studies of nonrandom cytoplasmic segregation during cellular differentiation. Spermatocytes separate from an anuclear cytoplasmic core and undergo two sequential divisions. During the second division, intracellular organelles segregate specifically to spermatids as they bud from an anuclear residual body. We have applied patch-clamp techniques in order to investigate membrane protein distribution during these asymmetric divisions. We show that membrane components, as assayed by voltage-dependent ion channel activity, follow a specific distribution pattern during sperm development. Several voltage-sensitive ion channel activities are observed in spermatocytes and residual bodies, but only a single-channel type can be detected in spermatids, indicating that other channel activities are excluded from or inactivated within these cells as they form. The channel that is observed in spermatids is an inward-rectifying chloride channel (Clir), as indicated by its sensitivity to chloride channel inhibitors and Cl-dependent shifts in its conductance. Treatment of spermatids with Cl channel blockers induce their differentiation into spermatozoa, suggesting that Clir plays a role during this developmental step. These studies are the first application of patch-clamp electrophysiology to C. elegans development.

The universal signal for egg activation at fertilization is a rise in cytoplasmic Ca2+ with defined spatial and temporal kinetics. Mammalian and amphibian eggs acquire the ability to produce such Ca2+ signals during a maturation period that precedes fertilization and encompasses resumption of meiosis and progression to metaphase II. In Xenopus, immature oocytes produce fast, saltatory Ca2+ waves that can be oscillatory in nature in response to IP3. In contrast, mature eggs produce a single continuous, sweeping Ca2+ wave in response to IP3 or sperm fusion. The mechanisms mediating the differentiation of Ca2+ signaling during oocyte maturation are not well understood. Here, I characterized elementary Ca2+ release events (Ca2+ puffs) in oocytes and eggs and show that the sensitivity of IP3-dependent Ca2+ release is greatly enhanced during oocyte maturation. Furthermore, Ca2+ puffs in eggs have a larger spatial fingerprint, yet are short lived compared to oocyte puffs. Most interestingly, Ca2+ puffs cluster during oocyte maturation resulting in a continuum of Ca2+ release sites over space in eggs. These changes in the spatial distribution of elementary Ca2+ release events during oocyte maturation explain the continuous nature and slower speed of the fertilization Ca2+ wave. [Copyright &y& Elsevier]

Abstract: A transient increase in intracellular Ca2+ is the universal signal for egg activation at fertilization. Eggs acquire the ability to mount the specialized fertilization-specific Ca2+ signal during oocyte maturation. The first Ca2+ transient following sperm entry in vertebrate eggs has a slow rising phase followed by a sustained plateau. The molecular determinants of the sustained plateau are poorly understood. We have recently shown that a critical determinant of Ca2+ signaling differentiation during oocyte maturation is internalization of the plasma membrane calcium ATPase (PMCA). PMCA internalization is representative of endocytosis of several integral membrane proteins during oocyte maturation, a requisite process for early embryogenesis. Here we investigate the mechanisms regulating PMCA internalization. To track PMCA trafficking in live cells we cloned a full-length cDNA of Xenopus PMCA1, and show that GFP-tagged PMCA traffics in a similar fashion to endogenous PMCA. Functional data show that MPF activation during oocyte maturation is required for full PMCA internalization. Pharmacological and co-localization studies argue that PMCA is internalized through a lipid raft endocytic pathway. Deletion analysis reveal a requirement for the N-terminal cytoplasmic domain for efficient internalization. Together these studies define the mechanistic requirements for PMCA internalization during oocyte maturation. [Copyright &y& Elsevier]