Oocyte–Targeted Deletion Reveals That Hsp90b1 Is Needed for the Completion of First Mitosis in Mouse Zygotes

Hsp90b1 is an endoplasmic reticulum (ER) chaperone (also named Grp94, ERp99, gp96,Targ2, Tra-1, Tra1, Hspc4) (MGI:98817) contributing with Hspa5 (also named Grp78, BIP) (MGI:95835) to protein folding in ER compartment. Besides its high protein expression in mouse oocytes, little is known about Hsp90b1 during the transition from oocyte-to-embryo. Because the constitutive knockout of Hsp90b1 is responsible for peri-implantation embryonic lethality, it was not yet known whether Hsp90b1 is a functionally important maternal factor.To circumvent embryonic lethality, we established an oocyte-specific conditional knockout line taking advantage of the more recently created floxed Hsp90b1 line (Hsp90b1(flox), MGI:3700023) in combination with the transgenic mouse line expressing the cre recombinase under the control of zona pellucida 3 (ZP3) promoter (Zp3-cre, MGI:2176187). Altered expression of Hsp90b1 in growing oocytes provoked a limited, albeit significant reduction of the zona pellucida thickness but no obvious anomalies in follicular growth, meiotic maturation or fertilization. Interestingly, mutant zygotes obtained from oocytes lacking Hsp90b1 were unable to reach the 2-cell stage. They exhibited either a G2/M block or, more frequently an abnormal mitotic spindle leading to developmental arrest. Despite the fact that Hspa5 displayed a similar profile of expression as Hsp90b1, we found that HSPA5 and HSP90B1 did not fully colocalize in zygotes suggesting distinct function for the two chaperones. Consequently, even if HSPA5 was overexpressed in Hsp90b1 mutant embryos, it did not compensate for HSP90B1 deficiency. Finally, further characterization of ER compartment and cytoskeleton revealed a defective organization of the cytoplasmic region surrounding the mutant zygotic spindle.Our findings demonstrate that the maternal contribution of Hsp90b1 is critical for the development of murine zygotes. All together our data indicate that Hsp90b1 is involved in unique and specific aspects of the first mitosis, which brings together the maternal and paternal genomes on a single spindle

The Endoplasmic Reticulum Chaperone Protein GRP94 Is Required for Maintaining Hematopoietic Stem Cell Interactions with the Adult Bone Marrow Niche

Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is regulated by both intrinsic gene expression products and interactions with extrinsic factors in the HSC niche. GRP94, an endoplasmic reticulum chaperone, has been reported to be essential for the expression of specific integrins and to selectively regulate early T and B lymphopoiesis. In GRP94 deficient BM chimeras, multipotent hematopoietic progenitors persisted and even increased, however, the mechanism is not well understood. Here we employed a conditional knockout (KO) strategy to acutely eliminate GRP94 in the hematopoietic system. We observed an increase in HSCs and granulocyte-monocyte progenitors in the Grp94 KO BM, correlating with an increased number of colony forming units. Cell cycle analysis revealed that a loss of quiescence and an increase in proliferation led to an increase in Grp94 KO HSCs. This expansion of the HSC pool can be attributed to the impaired interaction of HSCs with the niche, evidenced by enhanced HSC mobilization and severely compromised homing and lodging ability of primitive hematopoietic cells. Transplanting wild-type (WT) hematopoietic cells into a GRP94 null microenvironment yielded a normal hematology profile and comparable numbers of HSCs as compared to WT control, suggesting that GRP94 in HSCs, but not niche cells, is required for maintaining HSC homeostasis. Investigating this, we further determined that there was a near complete loss of integrin α4 expression on the cell surface of Grp94 KO HSCs, which showed impaired binding with fibronectin, an extracellular matrix molecule known to play a role in mediating HSC-niche interactions. Furthermore, the Grp94 KO mice displayed altered myeloid and lymphoid differentiation. Collectively, our studies establish GRP94 as a novel cell intrinsic factor required to maintain the interaction of HSCs with their niche, and thus regulate their physiology

Estimating treated prevalence and service utilization rates: Assessing disparities in mental health

Abstract available at publisher's website.http://dx.doi.org/10.1002/sim.390

Issues in the Use of a Plant-Capture Method for Estimating the Size of the Street Dwelling Population

In 1990, the Census Bureau conducted two operations designed to include homeless persons in the census: an enumeration of the occupants of emergency shelters, and a late night enumeration of street sites identified by cities and census offices as places where homeless people congregate. To assess the street enumeration, the Census Bureau sponsored independent studies in which unobtrusive observers were stationed in a sample of street sites. The observers reported their observations and experience of the enumeration process in debriefing questionnaires filled out immediately after the 3 The observers reported their observations and experience of the enumeration process in debriefing questionnaires filled out immediately after the conclusion of the street enumeration. Data reported by the observers are applied here to fit a plant-capture model, which is an alternative to the classic capturerecapture method of estimating the size of a population. This method assumes that the plants (in ..

MiRP3 acts as an accessory subunit with the BK potassium channel.

MinK-related peptides (MiRPs) are single-span membrane proteins that assemble with specific voltage-gated K+ (Kv) channel alpha-subunits to establish gating kinetics, unitary conductance, expression level, and pharmacology of the mixed complex. MiRP3 (encoded by the KCNE4 gene) has been shown to alter the behavior of some Kv alpha-subunits in vitro but its natural partners and physiologic functions are unknown. Seeking in vivo partners for MiRP3, immunohistochemistry was used to localize its expression to a unique subcellular site, the apical membrane of renal intercalated cells, where one potassium channel type has been recorded, the calcium- and voltage-gated channel BK. Overlapping staining of these two proteins was found in rabbit intercalated cells, and MiRP3 and BK subunits expressed in tissue culture cells were found to form detergent-stable complexes. Electrophysiologic and biochemical evaluation showed MiRP3 to act on BK to reduce current density in two fashions: shifting the current-voltage relationship to more depolarized voltages in a calcium-dependent fashion ( approximately 10 mV at normal intracellular calcium levels) and accelerating degradation of MiRP3-BK complexes. The findings suggest a role for MiRP3 modulation of BK-dependent urinary potassium excretion

The resting and activated conformations of the voltage sensor of Ci-VSP from functional and solvent accessibility determinations

The voltage sensor domain (VSD) is responsible for electromechanical transduction in voltage-gated ion channels and enzymes. In all known VSDs, both architecture and voltage-sensing mechanism are conserved: the positive charged residues (R/K) on the fourth transmembrane segment S4 respond to the voltage change across the membrane, which trigger its own conformation change leading to the response of downstream domain. A wealth of biophysical information on voltage sensors in the last two decades has revealed one of the major functional states - “up” or activated state. However, the structure and functional properties of the “down” or resting state remains controversial. Here, we show electrophysiological and structural studies of the voltage sensor from Ciona intestinalis voltage sensitive phosphatase (Ci-VSP), that point to conformational transitions between the resting and activated conformations of the sensor. The voltage dependence of Ci-VSP mutants, analyzed by gating charge measurement in oocytes, show significant shift in their Q-V relationships along the voltage axis (R217E −60 mV, R217Q −20 mV, WT +60 mV, D136N +130 mV). At 0 mV, these mutants populate different functional states under biochemical conditions: WT and D136N mostly in the “down” state while R217E is mostly in the “up” state. A Ci-VSD biochemical preparation was developed for each of the four mutants and studied by site-directed spin labeling EPR (SDSL-EPR) methods in proteoliposomes. Mobility and accessibility information revealed the secondary structure of transmembrane segments and their positions relative to membrane and each other, suggesting the extend and direction of the motion of S4 between “up” and “down” states. These results are consistent with the down movement of S4 under hyperpolarization and render critical structural information, that allow us to propose a gating mechanism for Ci-VSD