Apical PAR protein caps orient the mitotic spindle in C. elegans early embryos.

During embryonic development, oriented cell divisions are important for patterned tissue growth and cell fate specification. Cell division orientation is controlled in part by asymmetrically localized polarity proteins, which establish functional domains of the cell membrane and interact with microtubule regulators to position the mitotic spindle. For example, in the 8-cell mouse embryo, apical polarity proteins form caps on the outside, contact-free surface of the embryo that position the mitotic spindle to execute asymmetric cell division. A similar radial or "inside-outside" polarity is established at an early stage in many other animal embryos, but in most cases, it remains unclear how inside-outside polarity is established and how it influences downstream cell behaviors. Here, we explore inside-outside polarity in C. elegans somatic blastomeres using spatiotemporally controlled protein degradation and live embryo imaging. We show that PAR polarity proteins, which form apical caps at the center of the contact-free membrane, localize dynamically during the cell cycle and contribute to spindle orientation and proper cell positioning. Surprisingly, isolated single blastomeres lacking cell contacts are able to break symmetry and form PAR-3/atypical protein kinase C (aPKC) caps. Polarity caps form independently of actomyosin flows and microtubules and can regulate spindle orientation in cooperation with the key polarity kinase aPKC. Together, our results reveal a role for apical polarity caps in regulating spindle orientation in symmetrically dividing cells and provide novel insights into how these structures are formed.

Apical PAR-3 caps orient the mitotic spindle in C. elegans early embryos.

During embryonic development, oriented cell divisions are important for patterned tissue growth and cell fate specification. Cell division orientation is controlled in part by asymmetrically localized polarity proteins, which establish functional domains of the cell membrane and interact with microtubule regulators to position the mitotic spindle. For example, in the 8-cell mouse embryo, apical polarity proteins form caps on the outside, contact-free surface of the embryo that position the mitotic spindle to execute asymmetric cell division. A similar radial or "inside-outside" polarity is established at an early stage in many other animal embryos, but in most cases it remains unclear how inside-outside polarity is established and how it influences downstream cell behaviors. Here, we explore inside-outside polarity in C. elegans somatic blastomeres using spatiotemporally controlled protein degradation and live embryo imaging. We show that PAR polarity proteins, which form apical caps at the center of the contact free membrane, localize dynamically during the cell cycle and contribute to spindle orientation and proper cell positioning. Surprisingly, apical PAR-3 can form polarity caps independently of actomyosin flows and the small GTPase CDC-42, and can regulate spindle orientation in cooperation with the key polarity kinase aPKC. Together, our results reveal a role for apical polarity caps in regulating spindle orientation in symmetrically dividing cells and provide novel insights into how these structures are formed.

Expression of a dominant negative PKA mutation in the kidney elicits a diabetes insipidus phenotype.

PKA plays a critical role in water excretion through regulation of the production and action of the antidiuretic hormone arginine vasopressin (AVP). The AVP prohormone is produced in the hypothalamus, where its transcription is regulated by cAMP. Once released into the circulation, AVP stimulates antidiuresis through activation of vasopressin 2 receptors in renal principal cells. Vasopressin 2 receptor activation increases cAMP and activates PKA, which, in turn, phosphorylates aquaporin (AQP)2, triggering apical membrane accumulation, increased collecting duct permeability, and water reabsorption. We used single-minded homolog 1 (Sim1)-Cre recombinase-mediated expression of a dominant negative PKA regulatory subunit (RIαB) to disrupt kinase activity in vivo and assess the role of PKA in fluid homeostasis. RIαB expression gave rise to marked polydipsia and polyuria; however, neither hypothalamic Avp mRNA expression nor urinary AVP levels were attenuated, indicating a primary physiological effect on the kidney. RIαB mice displayed a marked deficit in urinary concentrating ability and greatly reduced levels of AQP2 and phospho-AQP2. Dehydration induced Aqp2 mRNA in the kidney of both control and RIαB-expressing mice, but AQP2 protein levels were still reduced in RIαB-expressing mutants, and mice were unable to fully concentrate their urine and conserve water. We conclude that partial PKA inhibition in the kidney leads to posttranslational effects that reduce AQP2 protein levels and interfere with apical membrane localization. These findings demonstrate a distinct physiological role for PKA signaling in both short- and long-term regulation of AQP2 and characterize a novel mouse model of diabetes insipidus.

Cardiomyocytes from AKAP7 knockout mice respond normally to adrenergic stimulation.

Protein kinase A (PKA) is activated during sympathetic stimulation of the heart and phosphorylates key proteins involved in cardiac Ca(2+) handling, including the L-type Ca(2+) channel (Ca(V)1.2) and phospholamban (PLN). This results in acceleration and amplification of the beat-to-beat changes in cytosolic Ca(2+) in cardiomyocytes and, in turn, an increased rate and force of contraction. PKA is held in proximity to its substrates by protein scaffolds called A kinase anchoring proteins (AKAPs). It has been suggested that the short and long isoforms of AKAP7 (also called AKAP15/18) localize PKA in complexes with Ca(V)1.2 and PLN, respectively. We generated an AKAP7 KO mouse in which all isoforms were deleted and tested whether Ca(2+) current, intracellular Ca(2+) concentration, or Ca(2+) reuptake were impaired in isolated adult ventricular cardiomyocytes following stimulation with the ß-adrenergic agonist isoproterenol. KO cardiomyocytes responded normally to adrenergic stimulation, as measured by whole-cell patch clamp or a fluorescent intracellular Ca(2+) indicator. Phosphorylation of Ca(V)1.2 and PLN were also unaffected by genetic deletion of AKAP7. Immunoblot and RT-PCR revealed that only the long isoforms of AKAP7 were detectable in ventricular cardiomyocytes. The results indicate that AKAP7 is not required for regulation of Ca(2+) handling in mouse cardiomyocytes.

Cell-type specific expression of a dominant negative PKA mutation in mice.

We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any cell-type that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIα regulatory subunit (RIαB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIαB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIαB demonstrated a reduction in PKA activity and inhibition of cAMP-responsive gene expression. Mice expressing RIαB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIαB allele in vivo provides a novel system for the analysis of PKA function in physiology.

Mutations in AKAP5 disrupt dendritic signaling complexes and lead to electrophysiological and behavioral phenotypes in mice.

AKAP5 (also referred to as AKAP150 in rodents and AKAP79 in humans) is a scaffolding protein that is highly expressed in neurons and targets a variety of signaling molecules to dendritic membranes. AKAP5 interacts with PKA holoenzymes containing RIIalpha or RIIbeta as well as calcineurin (PP2B), PKC, calmodulin, adenylyl cyclase type V/VI, L-type calcium channels, and beta-adrenergic receptors. AKAP5 has also been shown to interact with members of the MAGUK family of PSD-scaffolding proteins including PSD95 and SAP97 and target signaling molecules to receptors and ion channels in the postsynaptic density (PSD). We created two lines of AKAP5 mutant mice: a knockout of AKAP5 (KO) and a mutant that lacks the PKA binding domain of AKAP5 (D36). We find that PKA is delocalized in both the hippocampus and striatum of KO and D36 mice indicating that other neural AKAPs cannot compensate for the loss of PKA binding to AKAP5. In AKAP5 mutant mice, a significant fraction of PKA becomes localized to dendritic shafts and this correlates with increased binding to microtubule associated protein-2 (MAP2). Electrophysiological and behavioral analysis demonstrated more severe deficits in both synaptic plasticity and operant learning in the D36 mice compared with the complete KO animals. Our results indicate that the targeting of calcineurin or other binding partners of AKAP5 in the absence of the balancing kinase, PKA, leads to a disruption of synaptic plasticity and results in learning and memory defects.

Cell-type-specific isolation of ribosome-associated mRNA from complex tissues.

Gene profiling techniques allow the assay of transcripts from organs, tissues, and cells with an unprecedented level of coverage. However, most of these approaches are still limited by the fact that organs and tissues are composed of multiple cell types that are each unique in their patterns of gene expression. To identify the transcriptome from a single cell type in a complex tissue, investigators have relied upon physical methods to separate cell types or in situ hybridization and immunohistochemistry. Here, we describe a strategy to rapidly and efficiently isolate ribosome-associated mRNA transcripts from any cell type in vivo. We have created a mouse line, called RiboTag, which carries an Rpl22 allele with a floxed wild-type C-terminal exon followed by an identical C-terminal exon that has three copies of the hemagglutinin (HA) epitope inserted before the stop codon. When the RiboTag mouse is crossed to a cell-type-specific Cre recombinase-expressing mouse, Cre recombinase activates the expression of epitope-tagged ribosomal protein RPL22(HA), which is incorporated into actively translating polyribosomes. Immunoprecipitation of polysomes with a monoclonal antibody against HA yields ribosome-associated mRNA transcripts from specific cell types. We demonstrate the application of this technique in brain using neuron-specific Cre recombinase-expressing mice and in testis using a Sertoli cell Cre recombinase-expressing mouse.

Tissue-specific PKA inhibition using a chemical genetic approach and its application to studies on sperm capacitation.

Studies on cAMP signaling and protein kinase A (PKA) function in vivo are limited by the lack of highly specific inhibitors that can be used in primary cell culture and whole animals. Previously we reported that a mutation in the ATP binding pocket of a catalytic subunit (Calpha) of PKA confers sensitivity to the pyrazolo[3,4-d]pyrimidine inhibitor, 1NM-PP1. We have now engineered the mouse Pkraca gene such that after Cre-mediated recombination in vivo, the CalphaM120A mutant protein is expressed and the wild-type Calpha is turned off. We demonstrate the utility of this approach by examining the requirement for PKA activity during capacitation of sperm from mice that express CalphaM120A mutant protein. For CalphaM120A sperm, 10 microM of 1NM-PP1 prevented PKA-dependent phosphorylation and the activation of motility that are both rapidly (<90 s) evoked by the HCO(3)(-) anion. A continuous (90 min) inhibition with 10 microM of 1NM-PP1 prevented the protein tyrosine phosphorylation of late-stage capacitation. Delayed application of 1NM-PP1 demonstrated that PKA activity was required for at least the initial 30 min of capacitation to produce subsequent protein tyrosine phosphorylation. Acute application of 1NM-PP1 rapidly slowed the accelerated beat of activated motility but did not affect the established waveform asymmetry of hyperactivated sperm. Our results demonstrate that PKA in CalphaM120A mutant sperm is rapidly and reversibly inhibited by 1NM-PP1 and that this blockade has selective and time-dependent effects on multiple aspects of capacitation. The conditional CalphaM120A-expressing mouse lines will be valuable tools for studying PKA function in vivo.

Resident enteric microbiota and CD8+ T cells shape the abundance of marginal zone B cells.

Since enteric microbial composition is a distinctive and stable individual trait, microbial heterogeneity may confer lifelong, non-genetic differences between individuals. Here we report that C57BL/6 mice bearing restricted flora microbiota, a distinct but diverse resident enteric microbial community, are numerically and functionally deficient in marginal zone (MZ) B cells. Surprisingly, MZ B-cell levels are minimally affected by germ-free conditions or null mutations of various TLR signaling molecules. In contrast, MZ B-cell depletion is exquisitely dependent on cytolytic CD8(+) T cells, and includes targeting of a cross-reactive microbial/endogenous MHC class 1B antigen. Thus, members of certain enteric microbial communities link with CD8(+) T cells as a previously unappreciated mechanism that shapes innate immunity dependent on innate-like B cells.

Objective changes in brow position, superior palpebral crease, peak angle of the eyebrow, and jowl surface area after volumetric radiofrequency treatments to half of the face.

BACKGROUND: Radiofrequency application through a proprietary device has recently been used for facial tissue tightening. Uniform volumetric heating of the dermis is created by passage of electrical current, while protection of the epidermis is maintained by concurrent cryogen cooling. OBJECTIVE: To objectively quantify the effectiveness of volumetric radiofrequency application on the face, we treated 10 patients on the left side of the face with radiofrequency and evaluated the changes in brow position, superior palpebral crease, angle of the eyebrow, and jowl surface area. METHODS: Uniform treatments were applied at 134 J/cm(2) to the left side of the forehead and 1 cm past midline, at 115 J/cm(2) to the left side of temple and cheeks, and at 97 J/cm(2) to the left side of the jaw line and inferior postauricular surface. Patients were evaluated at monthly intervals up to 3 months with digital photography. Morphologic changes were evaluated with the "measuring tool" and "angle tool" of the Mirror Suite imaging system for aligned frontal-view photographs (for eyebrow position, superior palpebral crease elevation, and eyebrow angle changes) and the "outline tool" for aligned oblique-view photographs (for jowl surface area changes). RESULTS: At the end of 3 months on the side that was treated, patients exhibited on average 4.3 mm of brow elevation and 1.9 mm of superior palpebral crease elevation along the midpupillary line and an average of 2.4 mm of brow elevation along the lateral canthal line. There was no significant improvement of brow elevation along the lateral canthal line on the contralateral side. The peak angle of the ipsilateral eyebrow became slightly more acute by an average of 4.5 degrees after treatments. Moreover, the jowls on the lower part of the face, displayed a mean decrease of 22.6% in surface area after treatments. The nontreated side displayed a lack of eyebrow angle and jowl surface area changes. CONCLUSIONS: The application of radiofrequency to the face provides quantifiable changes. The brow along the midpupillary line is elevated to a greater degree than the lateral brow. This is consistent with acute angle changes seen in the eyebrow. Improvements in the lower part of the face with radiofrequency application can be quantified by demonstrating a decrease jowl surface. Moreover, these measurement techniques can be useful tools for evaluating other treatment parameters with radiofrequency application.

Protein kinase C family functions in B-cell activation.

Members of the protein kinase C (PKC) family play important but distinct roles in B-cell activation, as demonstrated by emerging genetic and biochemical studies. PKCbeta is indispensable for B-cell antigen receptor (BCR)-induced NF-kappaB activation and B-cell survival. Recent evidence indicates that PKCbeta might regulate inhibitor of kappaB kinase (IKK) and NF-kappaB activation through interaction with the CARMA1/Bcl10/MALT signaling complex in BCR microdomains. By contrast, the novel PKC isoform PKCdelta is specifically required to maintain the tolerance of self-reactive B cells.

Signaling in transitional type 2 B cells is critical for peripheral B-cell development.

Splenic peripheral B-cell development and the events regulating this functionally significant but relatively poorly defined developmental process have become a major focus in recent studies in B-cell immunology. Following the exit from the bone marrow, peripheral B cells develop through transitional type 1 (T1) and transitional type 2 (T2) B-cell stages. Emerging data suggest that the T2 subset is the immediate precursor of the mature B-cell populations present in the spleen. In this review, we first elaborate on the evidence describing the unique properties of CD21hiCD24hiCD23hiIgMhiIgDhi T2 B cells. T2 cells uniquely activate a proliferative, pro-survival, and differentiation program in response to B-cell antigen receptor (BCR) engagement. The potential mechanisms leading to the differential BCR responsiveness of T1 versus T2 B cells are discussed. We also review evidence that distinguishes key BCR-dependent signaling pathways operative in T2 and mature B cells. These signaling cascades include a protein kinase Cbeta (PKCbeta)-dependent cell-survival pathway and a second PKCbeta-independent pathway essential for BCR-driven differentiation. Finally, we discuss recent intriguing results suggesting that the type of signal(s) encountered by T2 cells leads to their differential maturation toward the follicular mature versus marginal zone mature B-cell populations. These combined observations suggest important implications with regard to B-cell selection and tolerance, potential novel therapeutic targets for B-cell lymphomas, and how the intricate balance of commensal organisms and other microenvironmental signals interact to promote the generation of 'innate-like' versus adaptive effector B-cell populations.

Dysregulated TCL1 promotes multiple classes of mature B cell lymphoma.

The TCL1 protooncogene is overexpressed in many mature B cell lymphomas, especially from AIDS patients. To determine whether aberrant expression promotes B cell transformation, we generated a murine model in which a TCL1 transgene was overexpressed at similar levels in both B and T cells. Strikingly, transgenic mice developed Burkitt-like lymphoma (BLL) and diffuse large B cell lymphoma (DLBCL) with attendant Bcl-6 expression and mutated J(H) gene segments at a very high penetrance beginning at 4 months of age. In contrast, only one mouse developed a T cell malignancy at 15 months, consistent with a longer latency for transformation of T cells by TCL1. Activation of premalignant splenic B cells by means of B cell antigen receptor (BCR) engagement resulted in significantly increased proliferation and augmented AKT-dependent signaling, including increased S6 ribosomal protein phosphorylation. Transgenic spleen cells also survived longer than wild-type spleen cells in long-term culture. Together these data demonstrate that TCL1 is a powerful oncogene that, when overexpressed in both B and T cells, predominantly yields mature B cell lymphomas.

PKC-beta controls I kappa B kinase lipid raft recruitment and activation in response to BCR signaling.

NF-kappa B signaling is required for the maintenance of normal B lymphocytes, whereas dysregulated NF-kappa B activation contributes to B cell lymphomas. The events that regulate NF-kappa B signaling in B lymphocytes are poorly defined. Here, we demonstrate that PKC-beta is specifically required for B cell receptor (BCR)-mediated NF-kappa B activation. B cells from protein kinase C-beta (PKC-beta)-deficient mice failed to recruit the I kappa B kinase (IKK) complex into lipid rafts, activate IKK, degrade I kappa B or up-regulate NF-kappa B-dependent survival signals. Inhibition of PKC-beta promoted cell death in B lymphomas characterized by exaggerated NF-kappa B activity. Together, these data define an essential role for PKC-beta in BCR survival signaling and highlight PKC-beta as a key therapeutic target for B-lineage malignancies.

Increased basal cAMP-dependent protein kinase activity inhibits the formation of mesoderm-derived structures in the developing mouse embryo.

A targeted disruption of the RIalpha isoform of protein kinase A (PKA) was created by using homologous recombination in embryonic stem cells. Unlike the other regulatory and catalytic subunits of PKA, RIalpha is the only isoform that is essential for early embryonic development. RIalpha homozygous mutant embryos fail to develop a functional heart tube at E8.5 and are resorbed at approximately E10.5. Mutant embryos show significant growth retardation and developmental delay compared with wild type littermates from E7.5 to E10.5. The anterior-posterior axis of RIalpha mutants is well developed, with a prominent head structure but a reduced trunk. PKA activity measurements reveal an increased basal PKA activity in these embryos. Brachyury mRNA expression in the primitive streak of RIalpha mutants is significantly reduced, consistent with later deficits in axial, paraxial, and lateral plate mesodermal derivatives. This defect in the production and migration of mesoderm can be completely rescued by crossing RIalpha mutants to mice carrying a targeted disruption in the Calpha catalytic subunit, demonstrating that unregulated PKA activity rather than a specific loss of RIalpha is responsible for the phenotype. Primary embryonic fibroblasts from RIalpha mutant embryos display an abnormal cytoskeleton and an altered ability to migrate in cell culture. Our results demonstrate that unregulated PKA activity negatively affects growth factor-mediated mesoderm formation during early mouse development.

Mutation of the Calpha subunit of PKA leads to growth retardation and sperm dysfunction.

The intracellular second messenger cAMP affects cell physiology by directly interacting with effector molecules that include cyclic nucleotide-gated ion channels, cAMP-regulated G protein exchange factors, and cAMP-dependent protein kinases (PKA). Two catalytic subunits, Calpha and Cbeta, are expressed in the mouse and mediate the effects of PKA. We generated a null mutation in the major catalytic subunit of PKA, Calpha, and observed early postnatal lethality in the majority of Calpha knockout mice. Surprisingly, a small percentage of Calpha knockout mice, although runted, survived to adulthood. This growth retardation was not due to decreased GH production but did correlate with a reduction in IGF-I mRNA in the liver and diminished production of the major urinary proteins in kidney. The survival of Calpha knockout mice after birth is dependent on the genetic background as well as environmental factors, but sufficient adult animals were obtained to characterize the mutants. In these animals, compensatory increases in Cbeta levels occurred in brain whereas many tissues, including skeletal muscle, heart, and sperm, contained less than 10% of the normal PKA activity. Analysis of sperm in Calpha knockout males revealed that spermatogenesis progressed normally but that mature sperm had defective forward motility.

Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development.

Signaling through the Ag receptor is required for peripheral B lymphocyte maturation and maintenance. Defects in components of the B cell receptor (BCR) signalosome result in developmental blocks at the transition from immature (heat-stable Ag (HSA)(high)) to mature (HSA(low)) B cells. Recent studies have subdivided the immature, or transitional, splenic B cells into two subsets, transitional 1 (T1) and transitional 2 (T2) cells. T1 and T2 cells express distinct surface markers and are located in distinct anatomic locations. In this report, we evaluated the BCR signaling capacity of T1 and T2 B cell subsets. In response to BCR engagement, T2 cells rapidly entered cell cycle and resisted cell death. In contrast, T1 cells did not proliferate and instead died after BCR stimulation. Correlating with these results, T2 cells robustly induced expression of the cell cycle regulator cyclin D2 and the antiapoptotic factors A1/Bfl-1 and Bcl-x(L) and exhibited activation of Akt. In contrast, T1 cells failed to up-regulate these markers. BCR stimulation of T2 cells also led to down-regulation of CD21 and CD24 (HSA) expression, resulting in a mature B cell phenotype. In addition, T2 cells from Bruton's tyrosine kinase-deficient Xid mice failed to generate these proliferative and survival responses, suggesting a requirement for the BCR signalosome specifically at the T2 stage. Taken together, these data clearly demonstrate that T2 immature B cells comprise a discrete developmental subset that mediates BCR-dependent proliferative, prosurvival, and differentiation signals. Their distinct BCR-dependent responses suggest unique roles for T1 vs T2 cells in peripheral B cell selection.