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Open Access

SPE-8, a protein-tyrosine kinase, localizes to the spermatid cell membrane through interaction with other members of the SPE-8 group spermatid activation signaling pathway in C. elegans

  • Paul J Muhlrad1, 2,
  • Jessica N Clark3,
  • Ubaydah Nasri3,
  • Nicholas G Sullivan3 and
  • Craig W LaMunyon3Email author
BMC Genetics201415:83

DOI: 10.1186/1471-2156-15-83

Received: 9 April 2014

Accepted: 3 July 2014

Published: 14 July 2014

The SPE-8 group gene products transduce the signal for spermatid activation initiated by extracellular zinc in C. elegans. Mutations in the spe-8 group genes result in hermaphrodite-derived spermatids that cannot activate to crawling spermatozoa, although spermatids from mutant males activate through a pathway induced by extracellular TRY-5 protease present in male seminal fluid.

Here, we identify SPE-8 as a member of a large family of sperm-expressed non-receptor-like protein-tyrosine kinases. A rescuing SPE-8::GFP translational fusion reporter localizes to the plasma membrane in all spermatogenic cells from the primary spermatocyte stage through spermatids. Once spermatids become activated to spermatozoa, the reporter moves from the plasma membrane to the cytoplasm. Mutations in the spe-8 group genes spe-12, spe-19, and spe-27 disrupt localization of the reporter to the plasma membrane, while localization appears near normal in a spe-29 mutant background.

These results suggest that the SPE-8 group proteins form a functional complex localized at the plasma membrane, and that SPE-8 is correctly positioned only when all members of the SPE-8 group are present, with the possible exception of SPE-29. Further, SPE-8 is released from the membrane when the activation signal is transduced into the spermatid.

Caenorhabditis elegans Spermatogenesis Sperm activation spe-8 Signal transduction

The timing of sperm activation requires precise regulation. Activating too early wastes stored resources, and delayed activation put the sperm at a competitive disadvantage. Sperm activation, and spermatogenesis as a whole, have emerged as an important model system in the hermaphroditic nematode C. elegans[1]. Activation, also called spermiogenesis in C. elegans, is the final developmental transition in spermatogenesis when quiescent spherical spermatids undergo wholesale cellular reorganization, extend pseudopods, and begin to crawl. Two signaling molecules induce spermiogenesis: extracellular zinc [2] and the extracellular protease TRY-5 [3]. Both pathways are employed for male sperm activation, but only the zinc pathway is utilized in hermaphrodites for self-sperm activation [2]. The zinc signal is transduced into the spermatid via the products of at least five genes (spe-8, spe-12, spe-19, spe-27, and spe-29) named the spe-8 pathway for the first of the genes discovered [4]. Loss-of-function mutations in any of these genes result in an identical phenotype: hermaphrodites are self-sterile because their spermatids do not activate, while male mutants are fertile. Mutant hermaphrodites become self-fertile when inseminated by males because their self-spermatids are activated by TRY-5 in the seminal fluid. Given that spe-12, spe-19, and spe-29 encode membrane proteins [57], it has been proposed that a complex of the SPE-8 group proteins forms at the cell membrane to transduce the zinc-derived signal into the cell. Although spe-8 was the first gene of this group to be named, the molecular identity of spe-8 has not been published. Here, we report the identification of the spe-8 coding sequence, show that it encodes a member of a large family of sperm-expressed protein tyrosine kinases, and demonstrate that its gene product localizes to the membrane in a manner dependent upon the presence of the other spe-8 group gene products.

Identification of the spe-8coding sequence

Previous mapping placed spe-8 to the left end of Chromosome I, approximately 8.7 cM to the left of lin-17[4]. Subsequently, spe-8 was positioned extremely close (inseparable) to mex-3[8] (Figure 1A). Because essentially all spe and fer genes in C. elegans exhibit sperm-specific expression [9, 10], we reasoned that spe-8 expression is also specific to spermatogenesis. We surveyed microarray data comparing transcript abundance in hermaphrodites making only sperm [fem-3(q23gf)] with hermaphrodites producing only oocytes [fem-1(hc17ts)] [9, 10]. Of the predicted genes near mex-3, only two genes, F53G12.6 and F53G12.8, had a fem-3/fem-1 ratio >5, indicating sperm-specific expression (Figure 1A). F53G12.8 had already been eliminated as a candidate based on other results (Jeremy Nance, personal communication). We confirmed the sperm-specific expression of F53G12.6 by successful amplification of a PCR product specific from this gene in a fem-3 cDNA library but not in a fem-1 cDNA library (Figure 1B). Furthermore, a PCR product containing F53G12.6 plus its flanking DNA rescued spe-8(hc50) mutants (Figure 1C). Finally, we identified mutations that altered the F53G12.6 coding sequence in each of the spe-8 mutant strains (Figure 2). Taken together, these data indicate that F53G12.6 is the spe-8 gene.
Figure 1

Identification of the spe-8 coding sequence. A. The graph shows all the predicted genes on the far left end of Chromosome I, where spe-8 and mex-3 are known to be very close to one another. The Y axis plots the degree of sperm-specific expression measured as fem-3(q23)/fem-l(hcl7) microarray signal intensity [9, 10]. Two genes in the region, F53Gl2.6 and F53Gl2.8, appear spermatogenesis-specific. F53Gl2.8 had been previously eliminated as a candidate based on other results (Jeremy Nance, personal communication). B. Differential PCR analysis was used to confirn the sperm-specific expression of F53G12.6. PCR products were amplified from cDNA libraries derived either from fem-1(hcl7ts) hermaphrodites which produce only oocytes, or from fem-3(q23ts) hermaphrodites which produce only sperm. The PCR reactions were multiplexed for three possible products: (i) a 169 bp product from F53G12.6 cDNA, (ii) a 526 bp product from F25H8.1, a somatic gene included as a non-germline-specific control, and (iii) a 714 bp product from spe-12, included as a sperm-specific control. Both F53G12.6 and spe-12 products appear sperm specific by their appearance only in the fem-3 library, whereas the product from the somatic gene F25H8.1 was found in both libraries as expected. (PCR primers are given in Additional file 1). C. The results of transformation-rescue of spe-8(hc50) with wild-type PCR product containing F53G12.6 and its flanking sequence. Transformant worms produced a mean of 139.8 progeny (n = 12; SEM = 18.0) compared to a mean of 0.1 progeny (n = 15; SEM = 0.1) produced by their non-transformed siblings, indicating that F53G12.6 is spe-8.

Figure 2

spe-8 exonic structure and sequence alignment of its protein product. The exonic structure shown in A is supported by numerous EST sequences curated by WormBase ( The locations of mutations in the spe-8 gene were mapped onto (A) the exon structure and (B) a multiple alignment of SPE-8 from C. elegans and the top BLAST matches from other nematodes. The nucleotide alterations to codons are shown in A, and the amino acid changes are shown in B. Included in the alignment is the human c-Abl Tyrosine Kinase from structural analysis of the protein (Figure 3). The location of the SH2 domain is indicated by the red line, and the kinase domain is indicated by the blue line.

SPE-8 is a member of a family of C. eleganssperm proteins with non-receptor protein-tyrosine kinase and SH2 domains

The SPE-8 protein contains two domains, the first of which is an SH2 domain (Figure 2B, red line, and Figure 3). SH2 (Src homology 2) is a large family of protein domains that bind particular phosphorylated tyrosine residues on other proteins, and are key regulators of tyrosine phosphorylation signaling cascades [13]. Closely following the SH2 domain is a non-receptor protein-tyrosine kinase catalytic domain (Figure 2B, blue line, and Figure 3). SPE-8 is a member of the Fes/Fer protein-tyrosine kinase family [14, 15]. Alignment of the SPE-8 protein sequence with those of the best matching homologs from a BLASTP search against the nonredundant set of protein databases shows a high degree of conservation and the common presence of the N-terminal SH2 domain followed by a protein-tyrosine kinase domain, even among distantly related nematodes (Figure 2).
Figure 3

Predicted structural model of SPE-8. The model was predicted by TM-align [11] using iTasser [12], and is based on the crystal structure of the human c-Abl Tyrosine Kinase protein. The highly significant structural predictions for the SH2 and kinase domains are indicated by red and blue labeling, respectively. The structures of the N- and C-terminal domains were predicted by iTasser without known protein models and are labeled in gray. The amino acids affected by the spe-8 mutations are indicated in stick form and labeled purple. Also shown are the predicted kinase active site in green and a proline rich domain in gold.

A BLASTP of SPE-8 against C. elegans proteins returns a long list of paralogs (Additional file 2). For the 33 with a match significance ≤ 10−30, we examined two sources for information on sperm expression. One was the fem-3/fem-1 ratios from microarray data [9, 10], and the second was an analysis of the sperm transcriptome via RNA deep sequencing of sperm purified from him-5(e1490) males [16]. Twenty-nine of the proteins are upregulated in sperm in at least one of the two studies (Additional file 2). Such a large set of sperm-specific SPE-8 paralogs is not surprising, since spermatogenesis in C. elegans involves an over-abundance of kinases [9, 16]. All of the 29 sperm-expressed paralogs have the N-terminal SH2 domain followed by the kinase domain. Further, the majority (20) are located on Chromosome IV in a non-random distribution, with some located in two known sperm gene clusters [17] while others lie outside the clusters (Figure 4). The N-terminal ~100 residues of SPE-8 define the only region of the sequence that is unique among the sperm-specific paralogs, but the N-terminal domain matches that of all but one of the nematode SPE-8 homologs (Figure 2). Thus, the N-terminal domain likely provides functional specificity for SPE-8. Finally, each of the Caenorhabditis congeners shown in Figure 2B contains a large number of paralogs to its own SPE-8 homolog (data not shown).
Figure 4

The clustered arrangement of the sperm-expressed SPE-8 paralogs on Chromosome IV. The locations of the genes are based upon their nucleotide position, and those above the line are encoded on the positive strand, whereas those below the line are on the negative strand. Known spe genes are also shown. Chromosomal regions highlighted in red indicate known sperm gene clusters [17], and bold font indicates the two sperm genes on Chromosome IV with a sterile knockout phenotype.

We modeled the SPE-8 structure using the I-Tasser Protein Structure and Function Prediction Server [12]. The most significant structural alignments were to the human proteins c-Abl Tyrosine Kinase (Accession: 2FO0) and c-Src Tyrosine Kinase (Accession: 1FMK). While both proteins also included an SH3 domain very close to the N-Terminus, both proteins possess the SH2 and kinase domains and have crystal structures available [18, 19]. The c-Abl Tyrosine Kinase crystal structure was used for modeling SPE-8 with TM-align [11] because it had the most significant structural match (TM-score = 0.866; Figures 2 and 3). However, SPE-8 has N-terminal and C-terminal ends that were not predicted by c-Abl Tyrosine Kinase structure. We found no significant alignments or predictions for the N-terminal domain (AA’s 1–104) considered alone; however, it contains a stretch of seven sequential prolines that may mediate protein-protein interactions [20].

The effects of the various mutations in the spe-8 gene are illustrated in Figures 2 and 3. All of the missense mutations map to conserved residues (Figure 2B). A single mutation in the SH2 domain was recovered (hc134ts), and it is the only temperature sensitive allele. Mutations found in the predicted minor lobe of the kinase domain, the region responsible for ATP binding, are hc53 and hc79. Mutations found in the predicted major lobe of the kinase domain, the region responsible for recruiting target proteins to the active site, are hc40, hc50, and hc108. Because hc50 encodes a stop codon, we performed RT-PCR on RNA from hc50 mutants and found that the spe-8 transcript was present (Additional file 3). Therefore, hc50 eliminates the final 40 amino acid residues from the protein, including a small portion of the kinase domain and the entire C-terminal tail. In many protein kinases, a short C-terminal tail often contains a tyrosine residue, which can be phosphorylated, acting as a cis regulator of the active and inactive state of the protein [20]. The short C-terminus of SPE-8 does not have a tyrosine residue, so it is unclear whether this structure is autoregulatory.

A single mutation, hc85, originally attributed as an allele of spe-8[4], did not have a mutation in the spe-8 coding sequence. Our sequencing covered spe-8 from ~ 400 bp 5’ of start through ~200 bp 3’ of stop, with no alteration from wild-type. To test whether hc85 is actually an allele of spe-8, we performed a complementation test by crossing hc85 hermaphrodites and spe-8(hc50) males. The F1 were fertile, demonstrating that hc85 is not an allele of spe-8. The gene affected by hc85 remains to be determined.

SPE-8 localization

Using the Mos-SCI technique [21], we integrated SPE-8::GFP translational fusions into the ttTi5605 Mos1 transposon insertion on Chromosome II. One fusion had GFP on the N-terminus, and the other had GFP on the C-terminus. Both showed green fluorescence in the male germline. We crossed worms bearing the two fusions with spe-8(hc50) worms to determine if either reporter fusion rescued the spe-8 mutant phenotype. For the amino-terminal fusion, we observed 14 sterile worms out of 57 total F2, or 25%, which is expected from a monohybrid cross with no element present to rescue spe-8. Thus, the amino-terminal fusion did not rescue. However, for the carboxy-terminal fusion, only two of 69 F2 were sterile. This proportion, 2.9%, is significantly different from the 25% expected for no rescue (χ2 = 16.41; P = 0.00005), but it is similar to the percent expected if the fusion does rescue, where only 1/16 (6.2%) of the worms should be sterile. Thus, the carboxy-terminal fusion did rescue the spe-8(hc50) mutation, suggesting that the reporter provides an accurate portrait of SPE-8 localization. Therefore, we used the carboxy-terminal fusion to analyze SPE-8 localization.

SPE-8 localizes to the cell membrane during spermatogenesis. All cellular stages of spermatogenesis showed green fluorescence associated with the cell membrane (Figure 5, spe-8::GFP column). Unfortunately, developing sperm cells have autofluorescent internal structures. In comparison, control (non-reporter) developing sperm do not have green fluorescence associated with the cell membrane (Figure 5, N2 column). The primary spermatocytes expressing the reporter appear much more fluorescent even interior to the cell membrane than N2 controls, suggesting that SPE-8 may be accumulating in the cytosol and translocating to the membrane at this stage. By the secondary spermatocyte stage, the cytosol appears no more fluorescent than controls, but the plasma membrane is strongly fluorescent as the reporter has taken up residence there. At the budding spermatid stage a strong GFP signal comes from the residual body, where non-essential cellular components are disposed [22]. It is unclear if native SPE-8 enters the residual body, and an alternative possibility is that some of the fusion has degraded and segregated to the residual body. Finally, the membrane localization of GFP is most pronounced at the spermatid stage, when the cells await the activation signal.
Figure 5

Localization of a spe-8::GFP translational reporter in developing sperm. GFP was fused to the C-terminus of the spe-8 coding sequence and integrated into a Mos1 site on Chromosome II following the MosSCI technique. In the N2 column, cells from the unlabeled N2 strain are included as a negative control to illustrate the non-specific green autofluorescence found in C. elegans developing sperm. The spe-8::GFP column shows the green fluorescence in cells with the translational reporter. Note the arrows indicating membrane labeling not seen in the N2 controls. The remaining columns are from worms homozygous for the reporter construct insertion and a mutation in one of the spe-8 group genes. Membrane GFP localization was absent in spe-12(hc76), spe-19(ok3428), and spe-27(it132) mutant backgrounds, but some localization was present in a spe-29(it127) mutant background. Red fluorescence indicates DNA labeling by Hoechst 33342.

Our results also show that SPE-8 localization is dependent upon the other spe-8 group proteins. Localization is disrupted in all spe-8 group mutant backgrounds with the possible exception of spe-29 (Figure 5). In spe-12(hc76), spe-19(ok3428), and spe-27(it132) mutant backgrounds, there is no indication of reporter localization to the membrane. Indeed, the fluorescence seems to concentrate in the residual body, which might be indicative of degradation due to the loss of components of the mature activation complex. In a spe-29(it127) mutant background, reporter localization to the membrane is visible, but it is not as intense as in an otherwise wild-type background, perhaps as a consequence of the likelihood that the spe-29(it127) mutation does not result in a complete loss of function [6].

We also investigated SPE-8 localization in activated spermatozoa. In naturally activated spermatozoa from both males and hermaphrodites, the membrane localization found in spermatids disperses into the cytosol (Figure 6). Hermaphrodite self sperm activate exclusively through the SPE-8 group pathway signaled by extracellular zinc [2], so entry of SPE-8 into the cytoplasm is a normal consequence of zinc activation through the SPE-8 group. Male sperm utilize both the SPE-8 pathway and TRY-5 pathway [3]. While activated male sperm showed the same entry of SPE-8 into the cytoplasm, it is not clear whether TRY-5 alone releases SPE-8 from the membrane. What is clear is that TRY-5 activation is completely independent of the SPE-8 group: male spe-8 group mutants are fertile via the TRY-5 pathway, even though our results show that SPE-8 is mislocalized or absent in their sperm. SPE-8 entry into the cytoplasm also occurs in sperm activated proteolytically via Pronase in vitro (Figure 6). Pronase activation likely acts, at least in part, through the zinc-signaled SPE-8 group proteins, because spe-8 group mutant spermatids cannot be activated completely to spermatozoa by exposure to Pronase [23]. Interestingly, in vitro activation via triethanolamine (TEA) leaves SPE-8 localized to the membrane (Figure 6), which is not surprising given that TEA bypasses the activation pathway by mimicking a downstream spike in cytosolic pH [23].
Figure 6

Localization of the spe-8::GFP translational reporter in activated spermatozoa. Spermatids and spermatozoa were isolated from young adult hermaphrodites (first column). The GFP fluorescence has moved to the cytoplasm in the activated spermatozoa. A similar pattern was seen in spermatids and spermatozoa isolated from young adult males (second column). Spermatozoa activated with Pronase also have cytoplasmic GFP localization (third column, top), but when activated with triethanolamine (TEA), the GFP remains at the membrane (third column, bottom).

Here, we describe the identification, structure, and function of spe-8, and show that it is localized to the spermatid cell membrane but enters the cytoplasm during activation. spe-8 was the first of the five genes of the spe-8 group (spe-8, -12, -19, -27, and -29) to be identified by mutation. In contrast to the other four genes, whose sequences reveal little about their activity, the SPE-8 sequence suggests it belongs to one of the most prominent families of signaling molecules, the protein tyrosine kinases (PTKs) [24]. These proteins route information through cells by phosphorylating specific tyrosine residues on their substrate proteins, thereby altering their substrates' chemical activities and/or binding properties. The Src homology 2 (SH2) domain, a compact domain that recognizes and binds to specific phosphotyrosine-containing proteins, is a common protein module that is often associated with PTKs and is present in SPE-8. Beyond the strong sequence similarity to these two domains exhibited by SPE-8, the fact that all six of the identified mutations lie in conserved residues of the SH2 or PTK domain underscores the importance of these domains in SPE-8 function.

PTKs belong to two general classes, the receptor tyrosine kinases (RTKs) and the non-receptor tyrosine kinases (NRTKs) [6]. Whereas RTKs are typically bound to the plasma membrane through their own N-terminal structures, NRTKs may associate with membranes through associations of their N-terminal domains with co-receptor proteins. Based on overall sequence similarity, SPE-8 is most closely related to the Fer and Fes family of NRTKs [15]. Human Fes has both oncogenic and tumor suppressor roles, depending upon the type of tumor [14, 25], and Fer is widely expressed and activated by growth factors, primarily platelet derived growth factor (PDGF) [26]. Fer is involved in many processes including cell proliferation [27, 28], cell adhesion [29], cell migration [30], and even oocyte maturation [31]. Fer is also implicated in several cancers [3234]. Interestingly, a testis-specific alternatively-spliced isoform of Fer, FerT, has a distinct, shorter N-terminus than the full-length Fer protein and participates in acrosome formation through phosphorylation of actin remodeling proteins [35].

Both Fer and Fes have, in addition to their SH2 and kinase domains, several coiled-coil domains in their N-termini, which appear responsible for activation by oligomerization and subsequent trans-autophosphorylation of these proteins [18, 24]. NRTKs in general are found to localize in variable fashions either in the cytosol or bound to interior membranes via protein complexes [20]. The SPE-8 N-terminus is unique compared to all other members of the sperm expressed NRTK family in C. elegans. It is unclear if the N-terminus of SPE-8 is responsible for formation of dimers or oligomers, or if autophosphorylation occurs. However, we speculate that this structure may be responsible for protein-protein interactions, especially given its proline-rich region, many of which are implicated in protein-protein docking or in binding SH3 domains [20, 36, 37]. Perhaps the failure of our N-terminal GFP fusion to rescue the spe-8(hc50) mutation reflects the importance of the N-terminus in SPE-8 function. Unlike the unique N-terminus, the SPE-8 C-terminus matches those of some of the family members.

Whatever the function of the C- and N-terminal domains, it is clear that interactions among the SPE-8 group proteins act to localize or stabilize SPE-8 at the plasma membrane. Localization was disrupted in mutant backgrounds for spe-12, spe-19, and spe-27, and perhaps for spe-29. It is not surprising that SPE-12 and SPE-19 proteins are required for proper SPE-8 localization because both have transmembrane domains, and SPE-12 has been shown experimentally to reside in the cell membrane [5, 7]. However, the requirement of SPE-27 for proper SPE-8 localization is unexpected, given that SPE-27 is predicted to be cytosolic [38]. These results suggest SPE-27 may also be located at the plasma membrane. For SPE-29, the results were not clear. SPE-8 appeared to localize to the membrane in a mutant spe-29 background, but seemingly not as intensely as in a wild-type genomic background.

While SPE-8 associates with the membrane in developing sperm, its localization changes once the cells activate to spermatozoa. The protein appears to leave the membrane and become cytoplasmic in natively activated spermatozoa originating from both males and hermaphrodites. In both cases, the sperm are exposed to extracellular zinc, which acts through the SPE-8 group proteins to induce activation [2]. We have no method for observing SPE-8::GFP localization in sperm activated only through the TRY-5 pathway. It is not possible to activate sperm in vitro with TRY-5, and spe-8 group mutant male sperm are activated only by TRY-5, but SPE-8::GFP is mislocalized due to the spe-8 group mutation. In any case, SPE-8 is dispensable for TRY-5 activation. Not only does TRY-5 activation occur in spe-8 mutant backgrounds, but SPE-8 is also mislocalized in spe-12, spe-19, and spe-27 mutant backgrounds.

It is tempting to speculate as to the phosphorylation target of SPE-8. One possible target is SPE-6. SPE-6 has a predicted role maintaining the spermatid state by inhibiting activation until the SPE-8 group signal is transduced into the cell [39]. One hypothesis is that SPE-8 downregulates SPE-6 through tyrosine phosphorylation. SPE-6 protein exists in a perinuclear halo in spermatids (Jackson Peterson and Diane Shakes, personal communication), so the translocation of SPE-8 from the plasmalemma into the cytosol might enable interaction between the two proteins. Indeed, the NetPhos 2.0 Server [40] identifies four of the tyrosine residues in SPE-6 as high probability candidates for phosphorylation (data not shown). Another possible target is a C. elegans homolog of MPOP, the major sperm protein polymerization organizing protein identified in Ascaris suum[41, 42], although a C. elegans homolog has yet to be found. MPOP is an integral membrane protein distributed throughout the plasmalemma, but it is phosphorylated by a tyrosine kinase only on the leading edge of the pseudopod, where it nucleates MSP assembly to drive pseudopodial motility. However, SPE-8 is not likely the tyrosine kinase responsible for a putative C. elegans MPOP phosphorylation because SPE-8 appears restricted to the cell body in active spermatozoa (Figure 6).

SPE-8 shares both primary sequence and protein domain architecture similarity with at least 33 other predicted paralogs in C. elegans, 29 of which have been shown to be upregulated during spermatogenesis in at least one of two studies. We include genes identified as sperm-expressed in either study because each study has slight limitations: (i) the microarray studies [9, 10] did not include all genes in the genome and may suffer from cross-hybridization, and (ii) the RNA deep sequencing study [16] may miss early acting sperm genes expressed whose transcripts are degraded prior to cellular encapsulation at the primary spermatocyte stage. While the majority of these genes have not received experimental attention, 30 have knockout alleles available. Only four of those knockouts produced a sterile phenotype, with the rest having no phenotype (Additional file 2). RNAi reports suggest that some of the paralogs have somatic function, although even spe-8 has an RNAi phenotype of hypersensitivity to hypoxia [43]. Whether or not these paralogs have significant function in sperm remains unclear, but the fact that so few have phenotypes for the knockout alleles suggests that they have redundant function. The fact that so many kinases appear upregulated in sperm indicates that protein phosphorylation is a prominent regulatory mechanism during spermatogenesis. Such a conclusion is not surprising since (i) post-translational modification is the major regulatory mechanism available, owing to the cessation of gene expression at the spermatid stage, and (ii) protein phosphorylation is rapid, befitting the swift morphological transformations that take place during spermiogenesis.

Extracellular zinc is the signal for sperm activation transduced through the SPE-8 complex [2]. The zinc signal is transmitted across the membrane to SPE-8 tethered just inside the membrane. It is unclear how reception of the zinc signal releases SPE-8 from the membrane. Zinc is an important signaling molecule in numerous cell types. For instance, the activity of most cell types in the mammalian immune system is regulated by zinc [44]. Zinc alters sperm activity across many species, but its effect can be either positive or negative, depending upon the species (reviewed in [2]). Perhaps a more relevant example is the activation of p70 S6 kinase by extracellular zinc signaling in the progression of the cell cycle, with the possible involvement of intermediate NRTKs [45]. While the identities of all intermediate factors and their specific interactions are not known, extracellular zinc induced signaling involving kinases is widespread, and our work on SPE-8 helps in understanding how spatial localization is involved in the signaling events.

The spermatid activation signal initiated by extracellular zinc is transduced into the cell via the SPE-8 group proteins. Of the five proteins known to compose this pathway, only SPE-8 has a predicted functional domain, that being a non-receptor protein-tyrosine kinase. Three have predicted membrane domains (SPE-12, SPE-19, and SPE-29), and SPE-27 has no predicted domains. The association of the SPE-8 group proteins has been unclear. Our results suggest that the SPE-8 group proteins form a functional complex localized at the plasma membrane, and that SPE-8 is correctly positioned only when all members of the SPE-8 group are present, with the possible exception of SPE-29. Further, SPE-8 is released from the membrane when the activation signal is transduced into the spermatid. The identity of its phosphorylation target(s) is not yet clear, nor is whether SPE-8 kinase activity is up- or down-regulated by the activation signal. SPE-8 is also a member of a large family of sperm-expressed non-receptor protein tyrosine kinases in C. elegans, suggesting that the rapid changes associated with spermatogenesis – and spermatid activation in particular – are brought about, at least in part, through changes in phosphorylation.

Strains and general nematode methods

Worm strains were cultured by standard methods [46]. Some strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Others were either generated during the present work or were graciously provided by Samuel Ward and Steven W. L’Hernault. The following strains harbored spe-8 group gene mutations: BA785: spe-8(hc40) I, BA784: spe-8(hc50) I, BA786: spe-8(hc53) I, BA788: spe-8(hc79) I, BA801: spe-8(hc85) I (although we show here that hc85 is not an allele of spe-8), BA787: spe-8(hc108) I, BA797: spe-8(hc134ts) I, VC2971: spe-19(ok3428)/unc-51(e369) rol-9(sc148) V, BA963: spe-27(it132ts) IV, BA959: spe-29(it127) dpy-20(e1282) IV, BA783: spe-12(hc76) I. Translational GFP reporters were integrated into the ttTi5605 Mos1 insertion site in the strain EG4322: ttTi5605 II; unc-119(ed9) III. Sperm were activated in vitro using Pronase and triethanolamine following standard protocols [23].

Differential PCR analysis of spe-8

We used PCR to assess sperm-specific expression of spe-8. PCR was conducted on cDNA libraries constructed from either fem-1(hc17ts) or fem-3(q23ts) mutants (kindly provided by Harold Smith). Three different products were amplified using different primer pairs: (i) a spe-8 product (primers F53G12.6Lb and F53G12.6 L3'), (ii) a spe-12 product as a sperm upregulated positive control [7], and (iii) a product from F2SH8.1, which is expressed somatically (Jeremy Nance, personal communication). PCR primer sequences are listed in Additional file 1. PCR reaction conditions and cycling followed standard protocols [47].

Microinjection transformation rescue

We amplified a 4,273 bp fragment from wild-type worms containing the entire F53G12.6 gene, including 919 base pairs 5' of the start codon, and 592 base pairs 3' from the stop codon. We used Phusion High Fidelity DNA Polymerase (Thermo Scientific) for amplification following manufacturer’s protocols. For transformation rescue, a mixture containing 10 ng/μl of this PCR fragment, along with 100 ng/μl of the pCFJ104 plasmid containing the Pmyo-3::mCherry::unc-54 transcriptional fusion to track transformed progeny. The DNA mix was injected into young adult spe-8(hc50) mutant hermaphrodites that were then paired with males of the same mutant genotype. F1 and F2 hermaphrodite transformants were scored for self-fertility at 25°C using non-transformant sibs as controls.

Sequencing the spe-8mutants

To sequence the spe-8 mutant alleles, the spe-8 genomic region from the mutants was amplified by PCR either in two ~1,300 bp fragments (for mutations hc40, hc50, hc53, hc79, hc108, and hc134ts), or in a single large product corresponding to the rescuing fragment (for hc85). Phusion High Fidelity DNA Polymerase was used in amplification, and the fragments were sequenced by the Sanger method using the PCR primers and primers internal to the two fragments. All primer sequences are listed in Additional file 1.

RNA was extracted from a mixed age population of spe-8(hc50) worms. The worms were rinsed 4 times with M9 buffer, disrupted with a homogenizer in lysis buffer from the GeneJET™ RNA purification kit (Thermo Scientific), and the RNA was extracted following the manufacturer’s protocols. The RNA samples were treated with RQ1 DNase (Promega), and reverse transcription was performed with Maxima™ Reverse Transcriptase (Thermo Scientific) per manufacturer’s instructions with oligo (dT). We then multiplexed spe-8 specific primers (F53G12.6-F5 and F53G12.6-R5; see Additional file 1) with primers specific to act-2, the C. elegans ortholog of β-actin (see Additional file 1 for primer sequences) for amplification using Phusion High Fidelity DNA Polymerase (Thermo Scientific) per manufacturer’s protocols.

SPE-8 localization

Two translational GFP fusions with spe-8 were constructed following the Mos-SCI technique [21]. Both constructs included 838 bp of the sequence upstream of the start codon and 1039 bp downstream of the stop codon. The GFP coding sequence from plasmid pPD95.75 (Fire Lab C. elegans vector kit obtained from Addgene) was inserted either at the 5’ or the 3’ end of the genomic spe-8 sequence. The stop codon was eliminated from (i) GFP for the amino-terminal construct, or (ii) spe-8 for the carboxy-terminal construct. The various segments of the construct were amplified by PCR using Phusion High Fidelity DNA Polymerase (Thermo Scientific), and they were combined following the PCR fusion technique described by Hobert [48]. Primers used in the construction are listed in Additional file 1. The full-length constructs were cloned into the vector pCFJ151, which targets the ttTi5605 Mos1 transposon insertion on Chromosome II for homologous recombination. Following the Mos-SCI protocol [21], we recovered a homozygous integrated copy of each construct. The integrated reporters were crossed with spe-8(hc50) to determine if the reporters rescued spe-8 mutant sterility. The C-terminal reporter (zqIs8[spe-8::GFP]) rescued spe-8 mutant sterility (see Results) and was used in localization studies. We crossed the zqIs8[spe-8::GFP] translational reporter with mutations in the various spe-8 group genes to obtain strains that were homozygous for both zqIs8 and for the spe-8 group mutations. Microscopy was performed with a Nikon Eclipse Ti-S inverted microscope fitted for Differential Interference Contrast microscopy and epifluorescence. Localization was examined in the following strains: ZQ125: zqIs8 II; unc-119(ed3) III, ZQ126: spe-12(hc76) I; zqIs8 II, ZQ127: zqIs8 II; spe-29(it127) dpy-20(e1282ts) IV, ZQ128: zqIs8 II; spe-19(eb52) V, and ZQ137: zqIs8 II; spe-27(it132ts) IV.

We thank Andrew Singson for the continued urging to complete this project so that he could cite something other than WormBase for spe-8. Harold Smith, Jeremy Nance, and Samuel Ward provided helpful advice and discussion. Harold Smith also kindly provided the fem-1 and fem-3 cDNA libraries.

Authors’ Affiliations

Department of Molecular and Cellular Biology, University of Arizona
Department of Molecular, Cellular and Developmental Biology, Currently, University of Colorado Boulder
Department of Biological Science, California State Polytechnic University

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© Muhlrad et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.


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BM Gift – is this weird?

posted 3 years ago in Bridesmaids
  • poll: Ceramic egg holder as part of BM gift - Weird?
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    23 %
    Nope, I'd love that! : (47 votes)
    77 %
  • Post # 3
    309 posts
    Helper bee
    • Wedding: August 2015

    @KMoon5314:  super cute! I’ve never seen egg holders used like that but I’m pretty sure I need one now. I say if they liked it and they knew what it was for, do it!!

    Post # 4
    8478 posts
    Bumble Beekeeper
    • Wedding: April 2013

    @KMoon5314:  I want one, these look like awesome storage for beads/small crafts.

    Post # 5
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    • Wedding: March 2015

    Love it!!! And I think they would too. It is a great idea.

    Post # 6
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    • Wedding: September 2013

    i love it also

    Post # 11
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    Busy bee
    • Wedding: July 2014

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    I really like it! I would totally make use of that if I had one.

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    Helper bee
    • Wedding: October 2014

    LOVE IT! I checked out the Amazon link and it looks like it has a ton of uses too! So even if they didn’t want to use it as a jewelry holder it could be a cute option for storing eggs in your fridge or  for serving deviled eggs or other appetizers. What a cool idea.

    Post # 15
    2000 posts
    Buzzing bee
    • Wedding: October 2015

    @KMoon5314:  I LOVE that! I’d be over the moon with a gift like that. I’ve never even seen ceramic egg holders before, they’re really pretty.

    I’m sure your BMs will love their gift too, especially since they already told you they liked yours.  What a great idea! 🙂

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Tuesday, January 26, 2016

Bear with Leo in THE REVENANT

Back in 1972, Robert Redford went all scraggly and played a mountain man called JEREMIAH JOHNSON.  He was a deserter from war and in a hostile environment.  He battles the elements and fights Indians to remake his life alone in the wilderness.  When I watched Leonardo DiCaprio as the frontiersman in the film that brought him another Best Actor Oscar nomination, I felt like THE REVENANT could've been called WHAT'S EATING JEREMIAH JOHNSON?  He's battling the elements, there were some warrior Indians, he's got very little dialogue and he's all scraggly.  And, yes, he does deserve his current Oscar nomination.
 First of all, what is a revenant?  It's a person who has returned after death or a long absence.

Leonardo DiCaprio gives an outstanding performance as frontiersman Hugh Glass.  Director Alejandro Gonzalez Iñárritu gives an outstanding performance as director Terrence Malick (The Thin Red Line, The New World, The Tree of Life).  This is an extremely mystical film. It poses philosophical questions about survival and revenge and what the need for revenge does to one's spirit.  There's a bear attack scene that's one of the brutal and exciting scenes I saw in a 2015 film.  How that was shot, I don't want to know. I lost myself in the movie-making magic of that sequence.  There's a slight sexual overtone to that wild animal scene.  There's a moment or two when you can't tell whether it's an attack or a first date that went quite well.  A memorable movie scene.
ABC News and Good Morning America entertainment correspondent Chris Connelly was in place on GMA when the nominations came out.  He felt that the Oscar nomination for British actor Tom Hardy was a surprise.  I disagree.  But I've disagreed with Connelly about an Alejandro Gonzalez Iñárritu film before this one.  Hardy is one flat-out brilliant actor.  Watch him in The Revenant as the villain, the man who wants to kill Hugh -- finish him off and leave the body.  Hardy sounds just like a character plucked right out of a classic John Ford western.
 Now rent Bronson, an inventive psychological biopic drama about a notorious and highly violent British prisoner who changes his name to Charles Bronson.  He came from a socially upright middle-class family but wound up spending most of his adult life in solitary confinement behind bars.  Hardy is absolutely fascinating, frightening and occasionally funny in Bronson.  He burns up the screen.  He should've been a Best Actor Oscar nominee for his one-man tour-de-force in 2013's Locke.  He's the only person seen onscreen in that movie, playing a businessman on the road who takes phone calls while driving.  During the course of all those calls that reveal his personal drama, he decides which road to take in his life.  Hardy commands the screen for that entire film.  Critics raved about his performance.  Last year, critics practically did cartwheels over his leading man work in Mad Max:  Fury Road.  Couple that with last year's The Revenant .... and you've got Best Supporting Actor Oscar nominee Tom Hardy.  Here's what he looks like when he's not playing a grizzly man in the wild frontier.
 Iñárritu directed 2011's BIUTIFUL starring Javier Bardem as a family man who knows the mean streets of Barcelona and who has a psychic gift.  On GMA, a few minutes before the nominations were announced, Connolly felt that Bardem didn't have a chance at a nomination.  I wrote on Twitter that I felt Javier Bardem would be a Best Actor Oscar nominee for Biutiful.  Javier Bardem was a Best Actor Oscar nominee for Biutiful.

The director also guided Michael Keaton to a Best Actor Oscar nomination for Birdman, a backstage look at a movie actor attempting a comeback in a Broadway drama.  It took last year's Oscars for Best Director and Best Picture.  The Revenant got 12 Oscar nominations including Best Director and Best Picture.  Here's a trailer that's subtitled for foreign audiences.
The visuals are extraordinary.  The cinematography is rich and the acting is noteworthy.  But the movie is just too darn long.  The Revenant keeps you in the cold and snow for 2 hours and 36 minutes.  The director could have made all his points in 1 hour and 50 minutes.  I felt that same way about his 2006 drama, Babel.  It's a fine film but you do get that "Move on, you made this point already" feeling.  Babel clocked in at about 2 hours and 20 minutes.
 Admit it.  Leonardo DiCaprio has done some remarkable work over the years.  When I saw 1993's What's Eating Gilbert Grape?, I thought it was terrific that Hollywood found a special needs kid who could act.  That's how real young DiCaprio was in his performance.  Still, he's yet to take home some Hollywood gold.  Remember when he was Oprah's daytime guest to talk about his film, The Aviator?  Martin Scorcese's 2004 movie eventually brought Leo a Best Actor Oscar nomination.  Seated next to Oprah on her old daytime show stage, she gave her review of his performance this way:  She stood up and proclaimed, "Go get your Oscar, Leo!  Go get your Oscar, Leo!" And, of course, her Chicago audience erupted into wild applause.

And then, Ray, starring Jamie Foxx as blind singer/musician Ray Charles, opened.  Foxx got nominated for the Best Actor Oscar opposite DiCaprio for The Aviator.  Jamie took home the prize.  On her post-Oscars Hollywood special edition the day after the awards, Oprah hugged Oscar winner Jamie Foxx and said into his ear "I knew you'd win."

I really hoped Leo was watching Judge Judy instead of Oprah that day.  And I really hope he wins next month for The Revenant.  DiCaprio deserves the Hollywood gold.

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Proposed Blaine County Cancer Camp Draws Critics, Supporters

Feb 9, 2017

Camp Rainbow Gold serves kids and families struggling with cancer in Idaho. Currently, the nonprofit leases from two private camps in the Sawtooth Mountains, and camp officials want to build a permanent location near Ketchum to increase the number of kids who can attend.

But a vocal group of residents in the area have raised concerns about the proposed location of the new camp. They will get the chance to talk directly to county officials Thursday night when camp leaders present plans to obtain a Conditional Use Permit.

In letters submitted to the Blaine County Planning and Zoning Commission, opponents of the new location say campers would face potential wildfire danger and evacuation problems. Others claim they would spoil the remote beauty of the neighborhood by increasing traffic on a road shared with homeowners.

As of Wednesday afternoon, Blaine County had received 447 letters since July - both in support of and against the proposed location.

Find reporter Frankie Barnhill on Twitter @FABarnhill

Copyright 2017 Boise State Public Radio


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E-mail, Feeds, 'n' Stuff

Thursday, August 4, 2011

"Cottage"? Geez.

We blogged last evening about the scandal that the O's got brewing over the bargain rental of a developer's beach house to Portland's transportation director, Tom Miller. The property, owned by Beam Development chief Brad Malsin, was rented out to Miller for a sum that Miller said he couldn't recall, but Malsin said was $100 a night.

The O story called the place "Malsin's 450-square-foot cottage steps from the beach," but is that accurate? Look at this web page, particularly the photo on the right, of "Malsin Cottage." If that's the same place, it looks a lot bigger than 450 square feet from here -- as in, maybe 10 times that size. And it's so nice that they put it on a house tour.

Here's the Google street view: Link.

And here's the Google satellite view: Link.

Aside from Miler's preposterous assertion that he didn't remember the rent, there's something extremely funny going on here. How many other Portland officials have gotten the sweetheart deal on renting that place?

UPDATE, 1:32 p.m.: An alert reader has sent us to the Mapquest satellite view of the property, and it appears that yes, there are two ever-so-slightly detached buildings on the parcel, connected by a deck or walkway of some sort:

Perhaps the Miller party was confined to the back building. Perhaps. But it's still not going to have a rental value of only $100 a night on a June weekend.

Comments (43)

Gee, Brad has learned something! over the last few years since loosing out on the Burnside Bridgehead project!

The real shame is that a part-time blogger can conjure up more juicy tidbits in a couple of hours than full-time reporter for the state's "paper of record" who has probably been working on this for a week.

The REAL shame is that a dumb*ss commenter on this blog can find the assessor information in about 5 minutes, showing that the property is way bigger than 450 square feet.

Good link, Garage Wine. Clearly Miller is a weasel on this failure to report. Although page 3 of the "Annual Appraisal Report" link on the assessors link you provided does show an "Improvement 2" on the property of 432 "finished" square feet.

What bothers me most isn't the deed (?) itself but the complete lack of respect that's displayed. Not just here, with Miller, but everywhere in politics, from Kitzhaber/Hayes to Leonard/loos to Sam's envelope "loans". From no contract bids, 130mil bridge "planning" and systematic and deliberate fragmentation of the constituency, this the middle finger, folks.

During the first recall, I held a sign in front of City Hall telling anyone who could read that Adams was a Cheney-esque shill being marketed in a queer suit - and all the media could focus on was my homophobia!

84 paid PR hacks indeed. There's your job growth folks. Oh, and don't forget - you're # 1.

Facts are just so tiresome!

Garage Wine:
Great job!

I would add some blame going to the news editors if the Oregonian still has any. They're the ones that should be pushing their reporters. Perry White comes to mind.

Perhaps the positive outcome for all this will be twofold:
1. Oregonian will do a better job reporting; especially when they can be shown up by part-time bloggers
2. This house will show up on VRBO for $100/night so all can benefit!

Hi Jack,

As I wrote in the comments section of your earlier post, the property has two structures, a main house and a guest house/cabin/cottage.

Beth Slovic
The Oregonian

"Miler's preposterous assertion that he didn't remember the rent"

Because it was $0

Slovic, did you ask Miller or Maslin what Miller rented, house or cabin or both? Did you notice that even the assessors info has the cabin at 954 sq/ft and not 500? Reporting usually has followup questions, especially if you're going to step on Sam's toes or any of the other insider politicians/developers in town.

the property has two structures, a main house and a guest house/cabin/cottage.

Not on Google satellite view it doesn't. Unless the guest house is on Gulcana Avenue, it's one attached structure.

As I've stated here before, the thing with Portland corruption is they don't even realize they're doing it.

Everyone is a good progressive, on the same side. Everyone is on board with the same grand vision of transit-oriented redevelopment. They're all on the side of angels, so how could any of this stuff be wrong? It's just how it's been done here with zero push-back for decades and decades and decades.

Nothing will come of this story, unfortunately. Probably a one-off email from City HR reminding people to "be careful about the appearance of impropriety."

The real story, reported on once or twice, then forgotten, is how this Adams crony got into his current job in the first place. A very lucrative position for which he is completely unqualified.

Beth, the Oregonian's problem isn't just that it misses many of these stories, it's that you don't pursue the ones you find for more than a day.

How many other Portland officials have gotten the sweetheart deal on renting that place?

This is the point, not the size.

If I were an elected official, I would be careful to rent a place that had no political connections and pay the going rate. Would I be branded a fool for
turning down sweetheart deals?

Snards:...The real story, reported on once or twice, then forgotten, is how this Adams crony got into his current job in the first place. A very lucrative position for which he is completely unqualified...

That is the real story and there are many many more like it that the O prefers to not uncover.

The Oregonian has a lot of writers on the payroll. Most of them seem to spend their time rewriting PR releases. I wonder how many actually qualify as journalists.

Jack, if you use Google street view there *may* be two structures on the site after all. The sat view is a bit blurry and Google Maps doesn't quite nail the location. But if you go the the beach-end of the street and move east just a bit, you can street-view into the back of the lot -- it might very well be two structures. Not that this changes the debate...

If you look on satellite view, it's not ambiguous; there's one huge structure. The back is an add-on from the original house, which is probably 100 years old, but it's all connected. There is a small house northeast of the main house that faces north onto Gulcana; maybe that's part of the same parcel as what's pictured on this post as "Malsin Cottage," and maybe that's what Miller rented. Maybe. But even so, especially if it sleeps five or six people, it's worth more than a $100 a night in June. $100 that Miller can't remember paying.

On the county property tax map, the house on Gulcana is a separate parcel, owned by somebody else. Forget that theory. I don't see two buildings owned by Malsin.

Hi Jack,

You win; I shouldn't have used the word "structure." However, Malsin calls one part the "main house" and the other the "guest house." That's according to a public record.

Beth Slovic
The Oregonian

Someone should check with the City of Cannon Beach to see if (and what) is registered as a vacation rental? The City requies registration of vacation rentals.

I'd love to see the floor plan. It's hard to believe that the guests have to go outside to get to the kitchen.

I have to believe there's a second unit there--nobody would ever expect to get away with calling the structure pictured above 450 sq feet. Perhaps it is a housing unit above a garage or something?

It's probably an addition with a seperate entrance. A way of squeezing a second unit onto a lot that is supposed to have one unit. Something that Pdx planners love to do as well.

We're getting bogged down in details that are really beside the point.

Not really. If Miller was confined to 450 square feet, the size of the gift wasn't anywhere near as substantial as it would be if he had the run of that big beach house.

Portland Corruption is an insidious snake. It can be in a form like this beach house.

Or it can be like the situation at a meeting with Developers Mark Edlen and Homer Williams with CoP/PDC regarding SoWhat and the John Ross skyscraper. Mark promised a bunch of LEED energy components, making the surroundings GREEN. The staffs representing CoP/PDC complemented Mark/Homer and said that is why we enter into Public/Private Partnerships (without any public bidding) with developers that agree with Portland's agenda, how we want to develop the city.

Just do the Talk and you are on Portland's Goodwill Tour and considered The Favored One. Exchange of money can be diluted in many ways so that Ethic Committees can look the other way.

Well Jack, that raises a second question, of course, which is whether or not the 450 sq feet was where he stayed, or if it was a "you can stay in the cottage but feel free to use the main kitchen, media room, deck, etc." kind of deal.

Sheesh, at this point the easiest thing will be for Miller to say "hey, you know what, this looks bad, so I'll just go ahead and pay another $150 per night" to avoid the appearance of impropriety.

For me, it's not the money, but the fact that he knows the developer well enough to get in on this kind of deal.

I'd like to see somebody investigate which other government officials have stayed there.

I hope you have some free time, Jack. The O has already moved on to other stories and the other news outlets havent even touched this subject.

Maybe he actually stayed in a tent in the yard?
Did you try that one Miller?

Tom Miller's enjoying himself while he can; the city rumors are that (a)he's laughably incompetent, and (b)he's out the door with a change in administration.

I think Miller sees the writing on the wall. He's entirely out of his depth, and knows it.

The blatant cronyism exhibited by Miller's appointment as head of PBOT might be one reason that Adams saw polling that indicated he was toast. Maybe we should thank him. He'll be sweeping out skate parks a year and a half from now.

Mapquest has better defined topview. There does appear to be a sliver of space between the two buildings, but also a very cozy deck or porch common space. If you can get either of those beach properties for $100 a night out West in June that's where I'm taking my next vacation.

Newleaf, thanks for that excellent clarification.

"The blatant cronyism exhibited by Miller's appointment as head of PBOT might be one reason that Adams saw polling that indicated he was toast."

Cronyism that was illegal in Portland under the City Charter until Saltzman convinced city voters to give Commissioners the authority to hire anyone they wanted for these jobs. I wonder if he feels even a little bit of shame for this. By this one act Saltzman did more damage to the City than anyone other elected I know. OK Randy is worse.

If configured as you all have researched , it appears to be a fire-trap. If the second exit from each building is into a narrow passage it may be less-than-code....
Gotta wonder if he got permits to build [overbuild] in that fashion?

I rented a little place in pacific city (basically a little shack) probably not much bigger than 475 sqft, and paid around $125 a night, which was a great deal. And this was 3 years ago in the winter off season.

I would guess that the market rate for that place would be at least $300/night.

It's not *that* horribly underpriced--we got a full house (3 br, so way more than 450 sq feet) in Manzanita last year, last house on the road (so there was nothing between us and the beach except a small dune), and it was 225/night. So I'd say something around $175-$200/night would be market. As I said before, though, the money is not the problem as I see it-- the problem is that developers are just loaning out their houses to the heads of city departments.

There are in fact two separate houses on the property with different keys and alarms. Access to one does not mean access to the other. The guest cabin that Tom and his family stayed in is built above a garage. It is a tiny house with a sleeping loft built over the kitchen and bathroom - 450 SF or so - that can sleep 6 at the most (couch included), unless of course people are willing to sleep on the floor. We donated the house to a charity auction on several occasions...not sure if that means we should have registered the house with the local authorities or not, but I'm certain someone is willing to look into that for us.

Anyways, I thought I would clarify that they are in fact two separate structures connected by a common deck. No one has access to the main house unless visiting with Brad and Liz when they are guests are allowed to stay in the main house.

Did other PDC and CoP employees get the "friends and family" rate?

Do you still believe that renting to those who have the power to forgive (or lobby those in power to forgive) half million dollar loans doesn't raise any moral/legal concerns?

So how was the payment handled? Cash, check or card of some sort? There should be a record somewhere. Maybe I missed this part of the discussion.

Gee Whiz! Maybe I'll give Brad a cal and see if he will rent his "cottage" to me for a hundred bucks a night.
I feel slighted.

As a lawyer/blogger, I get
to be a member of:

In Vino Veritas

Locations, Spanish Red Wine
Locations, Argentinian Red Wine
La Antigua Clásico, Rioja 2011
Shatter, Grenache, Maury 2012
Argyle, Vintage Brut 2011
Abacela, Vintner's Blend #16 Abacela, Fiesta Tempranillo 2014
Benton Hill, Pinot Gris 2015
Primarius, Pinot Gris 2015
Januik, Merlot 2013
Napa Cellars, Cabernet 2013
J. Bookwalter, Protagonist 2012
LAN, Rioja Edicion Limitada 2011
Beaulieu, Cabernet, Rutherford 2009
Denada Cellars, Cabernet, Maipo Valley 2014
Marchigüe, Cabernet, Colchagua Valley 2013
Oberon, Cabernet 2014
Hedges, Red Mountain 2012
Balboa, Rose of Grenache 2015
Ontañón, Rioja Reserva 2015
Three Horse Ranch, Pinot Gris 2014
Archery Summit, Vireton Pinot Gris 2014
Nelms Road, Merlot 2013
Chateau Ste. Michelle, Pinot Gris 2014
Conn Creek, Cabernet, Napa 2012
Conn Creek, Cabernet, Napa 2013
Villa Maria, Sauvignon Blanc 2015
G3, Cabernet 2013
Chateau Smith, Cabernet, Washington State 2014
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Ca' del Baio Barbaresco Valgrande 2012
Goodfellow, Reserve Pinot Gris, Clover 2014
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Wente, Cabernet, Charles Wetmore 2011
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Selvapiana, Chianti Ruffina 2012
Joseph Carr, Cabernet 2012
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Road Work

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“Ranbir is extremely charming”: Ileana

Actress Ileana D’cruz who is starting her bollywood career with ‘Barfi’, is praying that the film gets off to a fantastic start. She also talks about her co-star Ranbir Kapoor and her role in the film.

The tollywood star said, “There is a deaf and mute boy called Barfi (Ranbir Kapoor) and an autistic girl, Jhilmil (Priyanka Chopra) but you get to see Barfi through my eyes.”

“I am the only person in the film who is not physically challenged. My character Shruti has led a very sheltered life. She holds back her emotions — until Barfi comes into her life and she realises how sad she is and starts enjoying life. She falls in love with him,” added the beautiful Illeana.

When asked if she enjoyed working with Ranbir, she said: “I am lucky to have Ranbir as my co-star in my first Bollywood film. Although I have sufficient amount of experience, it is still very important to have a co-star that you are comfortable with, who understands you, and who you can relate to, improvise and work with easily.”

“It was easy to work with him. He is very calm and relaxed and lovely. He is extremely charming not only on screen but off screen too. He is chivalrous, supportive, and willing to give of himself to get a better performance for someone else,” she added.

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Dialogue Promos Of Total Siyappa

Ali Zafar and Yami Gautam are all set to give you a nice time with their upcoming film. The film also stars Anupam Kher and Kiron Kher. Total Siyappa looks like a completely messed up film which will leave you laughing like a maniac. If you don’t believe what I’m saying then here you go, check out these promos. They speak volumes.

Dialogue Promo #1

This hilarious promos shows a little girl telling her aunt that she is pregnant. Her grand mom gets hyper and tells her not to play such games and play ludo instead. The video is funny and will leave you in splits of laughter, all thanks to Kiron Kher.

Dialogue Promo #2

This promo shows what all goes wrong when Ali Zafar comes to meet Yami Gautam’s parents. He is seen telling her that they should consider meeting some other day and not today. And Yami blatantly refuses.

Dialogue Promo #3

Here we see Kiron Kher asking Ali Zafar to heat up the soup in the kitchen. In the midst of chaos, the jar of soups falls out of the window hitting a man on his head. While Ali Zafar get worried, Yami tells him to chill as it wasn’t a murder but an accident.

Dialogue Promo #4

In this promo we see a hysterical Kiron Kher telling her daughter that she is not fine with a Pakistani. When her daughter says she loves that man, the mommy is quick to reply saying the day he gets three more wives, she will remember her mother’s words.

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Jennifer began her career in esthetics over 25 years ago with Lancôme Cosmetics.  A great opportunity to follow her skincare passion came when Philosophy Skincare recruited Jennifer to launch their skincare line for the Nordstrom retail accounts.
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