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T-complex 11 like 2 (TCP11L2) is a protein containing a serine-rich region in its N-terminal region. However, the function of TCP11L2 is unclear. Here, we showed that TCP11L2 expression gradually increased during muscle-derived satellite cell (MDSC) differentiation in vitro, reaching a peak on Day 3, which is the migration and fusion stage of MDSCs. Using CRISPR/dCas9 gene-editing technology to elevate or repress the expression of TCP11L2, we also showed that TCP11L2 promoted MDSC differentiation. Moreover, wound-healing assays showed that TCP11L2 promoted the migration of MDSCs during differentiation. Additionally, immunofluorescence analyses showed that TCP11L2 was mainly distributed around the microfilament and microtubules. Furthermore, the expression of TCP11L2 affected the expression of actin-related protein 2/3 (ARP2/3) complex. Co-immunoprecipitation assays and immunofluorescence analysis showed that TCP11L2 interacted with formin-like 2 (FMNL2). This protein promoted migration of bovine MDSCs by affecting the expression of ARP2/3. Finally, the activities of TCP11L2 during MDSC differentiation and migration were blocked when FMNL2 was inhibited. Taken together, our data established that TCP11L2 interacted with FMNL2 to promote MDSC migration and differentiation.

Mammalian spermatozoa employ calcium (Ca2+) and cyclic adenosine monophosphate (cAMP) signaling in generating flagellar beat. However, how sperm direct their movement towards the egg cells has remained elusive. Here we show that the Rho small G protein RAC1 plays an important role in controlling progressive motility, in particular average path velocity and linearity. Upon RAC1 inhibition of wild type sperm with the drug NSC23766, progressive movement is impaired. Moreover, sperm from mice homozygous for the genetically variant t-haplotype region (tw5/tw32), which are sterile, show strongly enhanced RAC1 activity in comparison to wild type (+/+) controls, and quickly become immotile in vitro. Sperm from heterozygous (t/+) males, on the other hand, display intermediate RAC1 activity, impaired progressive motility and transmission ratio distortion (TRD) in favor of t-sperm. We show that t/+-derived sperm consist of two subpopulations, highly progressive and less progressive. The majority of highly progressive sperm carry the t-haplotype, while most less progressive sperm contain the wild type (+) chromosome. Dosage-controlled RAC1 inhibition in t/+ sperm by NSC23766 rescues progressive movement of (+)-sperm in vitro, directly demonstrating that impairment of progressive motility in the latter is caused by enhanced RAC1 activity. The combined data show that RAC1 plays a pivotal role in controlling progressive motility in sperm, and that inappropriate, enhanced or reduced RAC1 activity interferes with sperm progressive movement. Differential RAC1 activity within a sperm population impairs the competitiveness of sperm cells expressing suboptimal RAC1 activity and thus their fertilization success, as demonstrated by t/+-derived sperm. In conjunction with t-haplotype triggered TRD, we propose that Rho GTPase signaling is essential for directing sperm towards the egg cells., Author summary Spermatozoa are highly specialized cells designed for one particular purpose: to swim towards the egg cells and fertilize them. Swimming is achieved by propelling rotation of the flagellum, a complex molecular motor. Many proteins building the flagellum have been identified, and mechanisms generating forward movement have been elucidated in some detail. However, how movement is directed towards the egg cells, is not well understood. In this study, we provide evidence that the Rho small GTPase RAC1 plays a pivotal role in sperm progressive movement. We show that inappropriately high or low RAC1 activity impairs progressive movement and the competitiveness of sperm within a sperm population. Sperm with balanced RAC1 activity therefore gain a selective advantage in reaching and fertilizing egg cells, as exemplified by the mouse t-haplotype. Our data highlight the importance of Rho signaling in controlling progressive motility, in sperm competition, non-Mendelian inheritance and male (in)fertility.

The proximal third of mouse chromosome 17 is known as the t-complex. The t-haplotype is a variant form of this region containing four tandem inversions compared with the wild-type t-complex, and thus recombination in heterozygotes of the t-haplotype is strongly suppressed along the entire t-complex region. Within this genetically locked t-haplotype, many mutations related to various interesting phenotypes (e.g., taillessness, transmission ratio distortion, recessive lethality) have accumulated, and many mouse geneticists have been attracted to t-haplotype research. Many recessive lethal mutations known as t-complex lethal mutations have been found, and detailed phenotypic analyses have revealed that the functions of t-lethal genes are related to important developmental events. Therefore, identification of the genes responsible for these lethal mutations may contribute to our understanding of the mechanisms of mammalian development. In this review, I introduce the phenotypes of t-lethal mutations and describe recent findings, including our results regarding the molecular identification of a t-lethal gene.

TCPl, the human homolog of the Tcp-1 locus in the mouse, which is part of the murine t complex and codes for an abundant testicular germ-cell protein, has been mapped within the human genome by in situ hybridization. Using a cDNA probe for TCPl, pB1.4hum, we assigned TCPl to human chromosome region 6q23→qter, with the most likely localization being 6q25→q27.

After implantation, pluripotent epiblasts are converted to embryonic ectoderm through cell-cell interactions that significantly change the transcriptional and epigenetic networks. An entree to understanding this vital developmental transition is the t(w5) mutation of the mouse t complex. This mutation produces highly specific defects in the embryonic ectoderm before gastrulation, leading to death of the embryonic ectoderm. Using a positional cloning approach, we have now identified the mutated gene, completing a decades-long search. The gene, vacuolar protein sorting 52 (Vps52), is a mouse homolog of yeast VPS52 that is involved in the retrograde trafficking of endosomes. Our data suggest that Vps52 acts in extraembryonic tissues to support the growth and differentiation of embryonic ectoderm via cell-cell interactions. It is also required in the formation of embryonic structures at a later stage of development, revealing hitherto unknown functions of Vps52 in the development of a multicellular organism.

Transmission ratio distortion (TRD) by the mouse t-haplotype, a variant region on chromosome 17, is a well-studied model of non-Mendelian inheritance. It is characterized by the high transmission ratio (up to 99%) of the t-haplotype from t/+ males to their offspring. TRD is achieved by the exquisite ability of the responder (Tcr) to trigger non-Mendelian inheritance of homologous chromosomes. Several distorters (Tcd1-Tcd4), which act cumulatively, together promote the high transmission ratio of Tcr and the t-haplotype. Molecularly, TRD is brought about by deregulation of Rho signaling pathways via the distorter products, which impair sperm motility, and the t-sperm specific rescue of sperm motility by the responder. The t-sperm thus can reach the egg cells faster than +-sperm and fertilize them. Previously we have shown that the responder function is accomplished by a dominant negative form of sperm motility kinase (SMOKTCR), while the distorter functions are accomplished by the Rho G protein regulators TAGAP, FGD2 and NME3 proposed to function in two oppositely acting pathways. Here we identify the RAC1-specific guanine nucleotide exchange factor TIAM2 as modifier of t-haplotype TRD. Tiam2 is expressed in two isoforms, the full-length (Tiam2l) and a short transcript (Tiam2s). Tiam2s expression from the t-allele is strongly increased compared to the wild-type allele. By transgenic approaches we show that Tiam2s enhances t-haplotype transmission, while Tiam2l has the opposite effect. Our data show that a single modifier locus can encode different gene products exerting opposite effects on a trait. They also suggest that the expression ratio of the isoforms determines if the outcome is an enhancing or a suppressive effect on the trait., Author summary Exceptions to Gregor Mendel’s rules of inheritance have attracted geneticists over many decades. One of the best-studied cases in mammals is the extremely high transmission rate (up to 99%) of a chromosomal variant in mouse, called the t-haplotype, from t/+ heterozygous males to their offspring. Previous studies have revealed that non-equal inheritance of the homologous chromosomes is due to motility differences between sperm containing the t-haplotype or the +-chromosome. Several regulators of Rho small G protein signaling additively contribute to the motility defects, while a mutant protein kinase is able to rescue sperm motility. However, it does so only in sperm containing the t-haplotype enabling them to reach the egg cells faster than the competing +-sperm. In this work we show that Tiam2, a gene known to be involved in cell motility control and tumor metastasis, is expressed in two isoforms, which exert opposite effects on t-haplotype transmission. The relative amounts of the two transcripts can either promote t-haplotype transmission or act oppositely. Our study reveals a novel type of quantitative trait locus, which can switch between a promoting and an inhibiting state.

Despite deleterious effects on individuals, the t haplotype is a selfish genetic element present in many house mouse populations. By distorting the transmission ratio, +/t males transmit the t haplotype to up to 90% of their offspring. However, t/t individuals perish in utero. Theoretical models based on these properties predict a much higher t frequency than observed, leading to the t paradox. Here, we use empirical field data and theoretical approaches to investigate whether polyandry is a female counterstrategy against the negative fitness consequences of such distorters. We found a significant decrease of the t frequency over a period of 5.5 years that cannot be explained by the effect of transmission ratio distortion and recessive lethals, despite significantly higher life expectancy of +/t females compared to +/+ females. We quantified life-history data and homozygous and heterozygous fitness effects. Population subdivision and inbreeding were excluded as evolutionary forces influencing the t system. The possible influence of polyandry on the t system was then investigated by applying a stochastic model to this situation. Simulations show that polyandry can explain the observed t dynamics, making it a biologically plausible explanation for low t frequencies in natural populations in general.

Lfc is a guanine nucleotide exchange factor (GEF) for Rho that demonstrates an unusual ability to associate with microtubules. While several phosphorylated residues have been detected in the Lfc polypeptide, the mechanism(s) by which phosphorylation regulates the exchange activity of Lfc remains unclear. We confirm that Lfc is a phosphorylated protein and demonstrate that 14-3-3 interacts directly and in a phosphorylation-dependent manner with Lfc. We identify AKAP121 as an Lfc-binding protein and show that Lfc is phosphorylated in an AKAP-dependent manner by protein kinase A (PKA). Forskolin treatment induced 14-3-3 binding to Lfc and suppressed the exchange activity of wild-type Lfc on RhoA. Importantly, a mutant of Lfc that is unable to associate with 14-3-3 proteins was resistant to inhibition by forskolin. Tctex-1, a dynein motor light chain, binds to Lfc in a competitive manner with 14-3-3.

The mechanisms that regulate symmetric, proliferative divisions versus asymmetric, neurogenic divisions of mammalian neural precursors are still not well understood. We found that Lfc (Arhgef2), a Rho-specific guanine nucleotide exchange factor that interacts with spindle microtubules, and its negative regulator Tctex-1 (Dynlt1) determine the genesis of neurons from precursors in the embryonic murine cortex. Specifically, genetic knockdown of Arhgef2 in cortical precursors either in culture or in vivo inhibited neurogenesis and maintained cells as cycling radial precursors. Conversely, genetic knockdown of Dynlt1 in radial precursors promoted neurogenesis and depleted cycling cortical precursors. Coincident silencing of these two genes indicated that Tctex-1 normally inhibits the genesis of neurons from radial precursors by antagonizing the proneurogenic actions of Lfc. Moreover, Lfc and Tctex-1 were required to determine the orientation of mitotic precursor cell divisions in vivo. Thus, Lfc and Tctex-1 interact to regulate cortical neurogenesis, potentially by regulating mitotic spindle orientation.

Tau-tubulin kinase-1 (TTBK1) is involved in phosphorylation of tau protein at specific Serine/Threonine residues found in paired helical filaments, suggesting its role in tauopathy pathogenesis. We found that TTBK1 levels were upregulated in brains of human Alzheimer' disease (AD) patients compared with age-matched non-AD controls. To understand the effects of TTBK1 activationin vivo, we developed transgenic mice harboring human full-length TTBK1 genomic DNA (TTBK1-Tg). Transgenic TTBK1 is highly expressed in subiculum and cortical pyramidal layers, and induces phosphorylated neurofilament aggregation. TTBK1-Tg mice show significant age-dependent memory impairment as determined by radial arm water maze test, which is associated with enhancement of tau and neurofilament phosphorylation, increased levels of p25 and p35, both activators of cyclin-dependent protein kinase 5 (CDK5), enhanced calpain I activity, and reduced levels of hippocampal NMDA receptor types 2B (NR2B) and D. Enhanced CDK5/p35 complex formation is strongly correlated with dissociation of F-actin from p35, suggesting the inhibitory mechanism of CDK5/p35 complex formation by F-actin. Expression of recombinant TTBK1 in primary mouse cortical neurons significantly downregulated NR2B in a CDK5- and calpain-dependent manner. These data suggest that TTBK1 in AD brain may be one of the underlying mechanisms inducing CDK5 and calpain activation, NR2B downregulation, and subsequent memory dysfunction.

Despite extensive data demonstrating that immature retroviral particle assembly can take place either at the plasma membrane or at a distinct location within the cytoplasm, targeting of viral precursor proteins to either assembly site still remains poorly understood. Biochemical data presented here suggest that Tctex-1, a light chain of the molecular motor dynein, is involved in the intracellular targeting of Mason–Pfizer monkey virus (M-PMV) polyproteins to the cytoplasmic assembly site. Comparison of the three-dimensional structures of M-PMV wild-type matrix protein (wt MA) with a single amino acid mutant (R55F), which redirects assembly from a cytoplasmic site to the plasma membrane, revealed different mutual orientations of their C- and N-terminal domains. This conformational change buries a putative intracellular targeting motif located between both domains in the hydrophobic pocket of the MA molecule, thereby preventing the interaction with cellular transport mechanisms.

Previous studies on the fourth inversion of the t complex, In17(4), suggest that loci near the center of this inversion have been subjected to segmental recombination during the past 1–2 million years. We have used a combination of PCR-based restriction site (PBR) analysis and DNA sequencing to perform a high-resolution analysis of a 2-million base pair (Mbp) segment in the middle of In17(4). We examined 21 restriction sites that are polymorphic between t haplotypes and their wild-type homologs, over nine distinct loci. In addition, we examined several other polymorphic sites through DNA sequence analysis of two of these nine loci. We analyzed several haplotypes in this way, including the “complete” t haplotypes t w2 , t 0 , t w32 , t w71 , and t w75 . We show that only t w32 is a true “complete” t haplotype; the remaining four t haplotypes have segments of wild-type DNA ranging from less than 100 bp to 2 Mbp. The sizes of these wild-type DNA segments are consistent with their being generated by gene-conversion events. The 2-Mbp segment is located in a region that may contain the t-complex distorter gene Tcd2. One of the nine loci examined in this study is Fgd2, a gene that has been proposed to encode Tcd2. Sequencing and PBR data show that at least a portion of the Fgd2 gene has been converted to the wild-type within t w71 and t w75 mice.

International audience; After their discovery in the first decades of the XX th century, pseudoalleles generated much interest among geneticists: they apparently violated the conception of the genome as a collection of independent genes elaborated by Thomas Morgan's group. Their history is rich, complex, and deserves more than one short contribution. I will focus on two issues: the way the phenomenon of pseudoallelism suggested that the genome was more than a simple addition of independent genes, and the connection established between the formation of pseudoalleles during evolution and their functional roles. I will successively consider the first explanations for the origin of pseudoalleles elaborated in the mid-1930s, the metabolic/developmental sequential model proposed by Ed Lewis in the 1950s, the disappointments encountered with the T-complex in the 1970s, and the fading of the previous models after the molecular characterization of the pseudoallelic gene complexes in the 1980s. Genomes are more than collections of genes, but their structures are the result of a complex evolutionary history that leaves no place for simplistic models.

Expression of T-complex testis expressed 5 (Tctex5), an orthologue of protein phosphatase-1 inhibitor-3 (PPP1R11), was enhanced in mouse testis and was also expressed in epididymis and spermatozoa. There were three transcripts of Tctex5 including one brain specific and two common transcripts dominant in mouse testis. Tctex5 protein isoforms (75, 52, 32, 25, and 14.3 kDa) were identified. Isoforms of 75 and 52 kDa were spermatogenic-specific and were found in protein fraction containing nuclei, mitochondria, and flagellum accessory, and also in protein fraction containing mainly membranes. Tctex5 was localized in nuclei of pachytene spermatocytes, round spermatocytes, cytoplasm of Sertoli cells in testis; cilia, secretion bodies and nuclei of epithelial cells and interstitium smooth muscle cells in epididymis; and head and principal piece of tail in epididymal spermatozoa. The results suggested that Tctex5 might be a specific protein phosphatase-1 inhibitor in sperm; various Tctex5 transcripts and isoforms and cellular locations imply its different roles in spermatogenesis. Nuclei-type isoforms (75 and 52 kDa) might take part in nucleus remodeling during spermatogenesis whilst membrane-type isoform (52 kDa) might be responsible for dephosphorylation of proteins during capacitation. The other isoforms might play general roles for all kinds of cell types.

The t haplotype is a variant form of the proximal third of mouse chromosome 17, which was isolated from wild mouse populations (Schimenti 2000; Lyon 2003). Heterozygous t/+ males preferentially (up to 99%) transmit the t chromosome to their offspring. Meiotic recombination between t-haplotype and wild-type chromosomes is strongly suppressed due to four large inversions, ensuring cotransmission of all factors involved in transmission ratio distortion (TRD) (Fig. 1A; Lyon 2003). However, rare recombinants have been isolated and utilized to identify and map four Distorter loci (t-complex distorters 1–4, Tcd1–4) and the single Responder (t-complex responder, Tcr) to chromosomal subregions of several megabases (Lyon 1984; Silver and Remis 1987). Genetic data demonstrated that Tcd loci act additively in enhancing the transmission rate of Tcr. It was suggested that Distorter genes have a harmful effect on sperm function, brought about by an action on the Responder, and that the wild-type allele of the Responder is more sensitive than the t form (Lyon 1986). Motility tests showed that part of the sperm derived from t/+ males displays altered beat frequency and impaired forward movement, while sperm from males carrying two t haplotypes becomes immotile quickly (Katz et al. 1979; Olds-Clarke and Johnson 1993). These and other data have led to the conclusion that Tcds compromise the motility of all sperm, while Tcr specifically rescues sperm carrying this gene, giving t sperm an advantage in reaching the oocytes. Figure 1. Fgd2 maps to the Tcd2 region. (A) Structure of a complete (tw5) and various partial (tx) t haplotypes used for mapping Fgd2. The Tcd2 region is defined as the segment of t chromatin that is present in th18 and exchanged for wild-type chromatin in tw18 ... A molecular dissection of the TRD phenomenon became possible through cloning of the Responder gene Smok1Tcr (Herrmann et al. 1999), which encodes a dominant-negative form of the protein kinase Smok1. The biochemical function of Tcr suggested that Tcd genes encode signaling molecules acting upstream of Smok1. In accordance with this model, we demonstrated that Tcd1a encodes the GTPase-activating protein Tagap1, a control factor of Rho G proteins (Bauer et al. 2005). This result prompted us to search the distal t-haplotype region for Distorter candidates, using the following criteria: The gene must (1) be located in the genomic interval comprising Tcd2, (2) encode a protein involved in signaling, (3) be expressed in testis, and (4) show alterations in the t haplotype as compared with the wild-type allele.

G protein-coupled receptors (GPCRs) are seven transmembrane receptors with an N-terminus in the extracellular region and C-terminus in the intracellular region. When an agonist binds to a GPCR, a signal is transduced into a cell through the activation of trimeric G proteins. Recently, it has been shown that the activities of GPCRs are regulated by multiple mechanisms. One of the mechanisms is regulation through the binding proteins to the carboxy (C)-terminus of GPCRs. In the present study, the binding partners for the C-terminus of the parathyroid hormone receptor (PTHR) and thromboxane A(2) receptor (TP) were searched for using yeast two-hybrid screening, and the functions of these proteins were investigated. We identified t-complex testis expressed-1 (Tctex-1) and 4.1G as associated proteins for the PTHR. Tctex-1 is one of the light chains of cytoplasmic dynein, which is a motor protein across microtubles. We found that Tctex-1 was involved in agonist-induced internalization of the PTHR. 4.1G, a cytoskeletal protein, facilitated the cell surface localization of the PTHR and augmented PHTR-mediated signal transduction. TPs consists of two splicing variants, TPalpha and TPbeta. As a result of yeast two-hybrid screening, two proteasomal proteins, proteasome activator PA28gamma and proteasome subunit alpha7, were identified as direct interacting proteins for TPbeta. TPbeta has a tendency to be retained in the intracellular compartment, probably due to its binding to proteasomes. We also demonstrated that TPalpha and TPbeta formed heterodimers, and the signal transduction through TPalpha was reduced by the formation of heterodimers. In conclusion, the proteins bound to GPCRs may regulate the intracellular traffic of GPCRs.

Computational analyses have identified the widespread occurrence of antisense transcripts in the human and the mouse genome. However, the structure and the origin of the majority of the antisense transcripts are unknown. The presence of antisense transcripts for 19 of 64 differentially expressed genes during mouse spermatogenesis was demonstrated with orientation-specific RT-PCR. These antisense transcripts were derived from a wide variety of origins, including processed sense transcripts, intronic and exonic sequences of a single gene or multiple genes, intergenic sequences, and pseudogenes. They underwent normal and alternative splicing, 5′ capping, and 3′ polyadenylation, similar to the sense transcripts. There were also antisense transcripts that were not capped and/or polyadenylated. The testicular levels of the sense transcripts were higher than those of the antisense transcripts in all cases, while the relative expression in nontesticular tissues was variable. Thus antisense transcripts have complex origins and structures and the sense and antisense transcripts can be regulated independently.

Neurofibrillary tangles form in a specific spatial and temporal pattern in Alzheimer's disease. Although tangle formation correlates with dementia and neuronal loss, it remains unknown whether neurofibrillary pathology causes cell death. Recently, a mouse model of tauopathy was developed that reversibly expresses human tau with the dementia-associated P301L mutation. This model (rTg4510) exhibits progressive behavioral deficits that are ameliorated with transgene suppression. Using quantitative analysis of PHF1 immunostaining and neuronal counts, we estimated neuron number and accumulation of neurofibrillary pathology in five brain regions. Accumulation of PHF1-positive tau in neurons appeared between 2.5 and 7 months of age in a region-specific manner and increased with age. Neuron loss was dramatic and region-specific in these mice, reaching over 80% loss in hippocampal area CA1 and dentate gyrus by 8.5 months. We observed regional dissociation of neuronal loss and accumulation of neurofibrillary pathology, because there was loss of neurons before neurofibrillary lesions appeared in the dentate gyrus and, conversely, neurofibrillary pathology appeared without major cell loss in the striatum. Finally, suppressing the transgene prevented further neuronal loss without removing or preventing additional accumulation of neurofibrillary pathology. Together, these results imply that neurofibrillary tangles do not necessarily lead to neuronal death.

Hyperactivation of the cyclin-dependent kinase 5 (cdk5), triggered by proteolytic conversion of its neuronal activator, p35, to a more potent byproduct, p25, has been implicated in Alzheimer's disease (AD), amyotrophic lateral sclerosis, and Niemann–Pick type C disease (NPC). This mechanism is thought to lead to the development of neuropathological hallmarks, i.e., hyperphosphorylated cytoskeletal proteins, neuronal inclusions, and neurodegeneration, that are common to all three diseases. This pathological ensemble is recapitulated in a single model, the npc-1 (npc−/−) mutant mouse. Previously, we showed that pharmacological cdk inhibitors dramatically reduced hyperphosphorylation, lesion formation, and locomotor defects innpc−/−mice, suggesting that cdk activity is required for NPC pathogenesis. Here, we used genetic ablation of thep35gene to examine the specific involvement of p35, p25, and hence cdk5 activation in NPC neuropathogenesis. We found that lack of p35/p25 does not slow the onset or progression or improve the neuropathology of NPC. Our results provide direct evidence that p35/p25-mediated cdk5 deregulation is not essential for NPC pathology and suggest that similar pathology in AD may also be cdk5 independent.

Cell–substrate contacts, called focal adhesions (FAs), are dynamic in rapidly moving cells. We show that supervillin (SV)—a peripheral membrane protein that binds myosin II and F-actin in such cells—negatively regulates stress fibers, FAs, and cell–substrate adhesion. The major FA regulatory sequence within SV (SV342-571) binds to the LIM domains of two proteins in the zyxin family, thyroid receptor–interacting protein 6 (TRIP6) and lipoma-preferred partner (LPP), but not to zyxin itself. SV and TRIP6 colocalize within large FAs, where TRIP6 may help recruit SV. RNAi-mediated decreases in either protein increase cell adhesion to fibronectin. TRIP6 partially rescues SV effects on stress fibers and FAs, apparently by mislocating SV away from FAs. Thus, SV interactions with TRIP6 at FAs promote loss of FA structure and function. SV and TRIP6 binding partners suggest several specific mechanisms through which the SV–TRIP6 interaction may regulate FA maturation and/or disassembly.

Chromosomal deletions can uncover haploinsufficient or imprinted regions of the genome. Previously, the haploinsufficient locus t haplolethal 1 (Thl1) was identified and localized to a 1.3-Mb region using overlapping deletions around the Sod2 and D17Leh94 loci of the mouse t complex on Chr 17. Germline chimeric mice, produced from embryonic stem (ES) cells containing radiation-induced deletions of the Thl1 locus, never produced viable deletion-bearing progeny when mated to C57BL/6J (B6) females. However, deletion-bearing offspring could be obtained by mating to females of other strains. In this article we describe a transgenic approach to narrow the critical region for Thl1. BAC clones were introduced into a deletion-bearing ES cell line and one was shown to rescue the Thl1 phenotype, reducing the critical region to 140 kb. Analysis of the gene content of this region suggests two strong Thl1 candidates, Pdcd2 and a novel SET domain-containing gene termed Tset1. A more detailed analysis using mice carrying overlapping deletions identified subregions that influence the phenotypic characteristics of Thl1 hemizygotes.

Heterozygosity for a t haplotype (t) in male mice results in distorted transmission (TRD) of the t-bearing chromosome 17 homolog to their offspring. However, homozygosity for t causes male sterility, thus limiting the spread of t through the population at large. The Ca(2+)-dependent sperm tail curvature phenotypes, "fishhook", where abnormally high levels of sperm exhibit sharp bends in the midpiece, and "curlicue", where motile sperm exhibit a chronic negative curving of the entire tail, have been tightly linked to t-associated male TRD and sterility traits, respectively. Genetic studies have indicated that homozygosity for the t allele of Dnahc8, an axonemal gamma-type dynein heavy chain (gammaDHC) gene, is partially responsible for expression of "curlicue"; however, its involvement in "fishhook"/TRD, if any, is unknown. Here we report that the major isoform of DNAHC8 is copiously expressed, carries an extended N-terminus and full-length C-terminus, and is stable and equally abundant in both testis and sperm from +/+ and t/t animals. By in silico analysis we also demonstrate that at least three of the seventeen DNAHC8(t) mutations at highly conserved positions in wild-type DHCs may be capable of substantially altering normal DNAHC8 function. Interestingly, DNAHC8 is confined to the principal piece of the sperm tail. The combined results of this study suggest possible mechanisms of DNAHC8(t) dysfunction and involvement in "curlicue", and support the hypothesis that "curlicue" is a multigenic phenomenon. They also demonstrate that the accelerated "fishhook" phenotype of sperm from +/t males is not directly linked to DNAHC8(t) dysfunction.

Retroviral tagging has been used extensively and successfully to identify genes implicated in cancer pathways. In order to find oncogenes implicated in T-cell leukemia, we used the highly leukemogenic radiation leukemia retrovirus VL3 (RadLV/VL3). We applied the inverted PCR technique to isolate and analyze sequences flanking proviral integrations in RadLV/VL3-induced T lymphomas. We found retroviral integrations in c- myc and Pim1 as already reported but we also identified for the first time Notch1 as a RadLV common integration site. More interestingly, we found a new RadLV common integration site that is situated on mouse chromosome X (XA4 region, bp 45091000). This site has also been reported as an SL3-3 and Moloney murine leukemia virus integration site, which strengthens its implication in murine leukemia virus-induced T lymphomas. This locus, named Kis2 (Kaplan Integration Site 2), was found rearranged in 11% of the tumors analyzed. In this article, we report not only the alteration of the Kis2 gene located nearby in response to RadLV integration but also the induction of the expression of Phf6 , situated about 250 kbp from the integration site. The Kis2 gene encodes five different alternatively spliced noncoding RNAs and the Phf6 gene codes for a 365-amino-acid protein which contains two plant homology domain fingers, recently implicated in the Börjeson-Forssman-Lehmann syndrome in humans. With the recent release of the mouse genome sequence, high-throughput retroviral tagging emerges as a powerful tool in the quest for oncogenes. It also allows the analysis of large DNA regions surrounding the integration locus.

Transmission ratio distortion in the mouse is caused by several t-complex distorters (Tcds) acting in trans on the t-complex responder (Tcr)1,2,3,4. Tcds additively affect the flagellar movement of all spermatozoa derived from t/+ males; sperm carrying Tcr are rescued, resulting in an advantage for t sperm in fertilization. Here we show that Tagap1, a GTPase-activating protein, can act as a distorter. Tagap1 maps to the Tcd1 interval and has four t loci, which encode altered proteins including a C-terminally truncated form. Overexpression of wild-type Tagap1 in sperm cells phenocopied Tcd function, whereas a loss-of-function Tagap1 allele reduced the transmission rate of the t6 haplotype. The combined data strongly suggest that the t loci of Tagap1 produce Tcd1a. Our results unravel the molecular nature of a Tcd and demonstrate the importance of small G proteins in transmission ratio distortion in the mouse.

Recombinant expression of actin in bacteria results in non-native species that aggregate into inclusion bodies. Actin is a folding substrate of TRiC, the chaperonin of the eukaryotic cytosol. By employing bacterial in vitro translation lysates supplemented with purified chaperones, we have found that TRiC is the only eukaryotic chaperone necessary for correct folding of newly translated actin. The actin thus produced binds deoxyribonuclease I and polymerizes into filaments, hallmarks of its native state. In contrast to its rapid folding in the eukaryotic cytosol, actin translated in TRiC-supplemented bacterial lysate folds with slower kinetics, resembling the kinetics upon refolding from denaturant. Lysate supplementation with the bacterial chaperonin GroEL/ES or the DnaK/DnaJ/GrpE chaperones leads to prevention of actin aggregation, yet fails to support its correct folding. This combination of in vitro bacterial translation and TRiC-assisted folding allows a detailed analysis of the mechanisms necessary for efficient actin folding in vivo. In addition, it provides a robust alternative for the production of substantial amounts of eukaryotic proteins that otherwise misfold or lead to cellular toxicity upon expression in heterologous hosts.

TcTex-1, one of three dynein light chains of the dynein motor complex, has been implicated in targeting and binding cargoes to cytoplasmic dynein for retrograde or apical transport. Interactions between TcTex-1 and a diverse set of proteins such as the dynein intermediate chain, Fyn, DOC2, FIP1, the poliovirus receptor, CD155, and the rhodopsin cytoplasmic tail have been reported; yet, despite the broad range of targets, a consensus binding sequence remains uncertain. Consequently, we have solved the crystal structure of the full-length Drosophila homolog of TcTex-1 to 1.7 A resolution using MAD phasing to gain insight into its function and target specificity. The structure is homodimeric with a domain swapping of beta-strand 2 and has a fold similar to the dynein light chain, LC8. Based on structural alignment, the TcTex-1 and LC8 sequences show no identity, although the root mean square deviation between secondary structural elements is less than 1.6 A. Moreover, the N terminus, which is equivalent to beta-strand 1 in LC8, is splayed out and binds to a crystallographic dimer as an anti-parallel beta-strand at the same position as the neuronal nitric-oxide synthase peptide in the LC8 complex. Similarity to LC8 and comparison to the LC8-neuronal nitricoxide synthase complex suggest that TcTex-1 binds its targets in a similar manner as LC8 and provides insight to the lack of strict sequence identity among the targets for TcTex-1.

In search for the genetic basis of hypertension, we applied an integrated genomic-transcriptomic approach to identify genes involved in the pathogenesis of hypertension in the Sabra rat model of salt-susceptibility. In the genomic arm of the project, we previously detected in male rats two salt-susceptibility QTLs on chromosome 1, SS1a ( D1Mgh2-D1Mit11 ; span 43.1 cM) and SS1b ( D1Mit11-D1Mit4 ; span 18 cM). In the transcriptomic arm, we studied differential gene expression in kidneys of SBH/y and SBN/y rats that had been fed regular diet or salt-loaded. We used the Affymetrix Rat Genome RAE230 GeneChip and probed >30 000 transcripts. The research algorithm called for an initial genome-wide screen for differentially expressed transcripts between the study groups. This step was followed by cluster analysis based on 2×2 ANOVA to identify transcripts that were of relevance specifically to salt-sensitivity and hypertension and to salt-resistance. The two arms of the project were integrated by identifying those differentially expressed transcripts that showed an allele-specific hypertensive effect on salt-loading and that mapped within the defined boundaries of the salt-susceptibility QTLs on chromosome 1. The differentially expressed transcripts were confirmed by RT-PCR. Of the 2933 genes annotated to rat chromosome 1, 1102 genes were identified within the boundaries of the two blood pressure QTLs. The microarray identified 2470 transcripts that were differentially expressed between the study groups. Cluster analysis identified genome-wide 192 genes that were relevant to salt-susceptibility and/or hypertension, 19 of which mapped to chromosome 1. Eight of these genes mapped within the boundaries of QTLs SS1a and SS1b . RT-PCR confirmed 7 genes, leaving TcTex1, Myadm, Lisch7, Axl-like, Fah, PRC1-like, and Serpinh1 . None of these genes has been implicated in hypertension before. These genes become henceforth targets for our continuing search for the genetic basis of hypertension.

Voltage-gated Ca(2+) channels (VGCCs) are important in regulating a variety of cellular functions in neurons. It remains poorly understood how VGCCs with different functions are sorted within neurons. Here we show that the t-complex testis-expressed 1 (tctex1) protein, a light-chain subunit of the dynein motor complex, interacts directly and selectively with N- and P/Q-type Ca(2+) channels, but not L-type Ca(2+) channels. The interaction is insensitive to Ca(2+). Overexpression in hippocampal neurons of a channel fragment containing the binding domain for tctex1 significantly decreases the surface expression of endogenous N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels, as determined by immunostaining. Furthermore, disruption of the tctex1-Ca(2+) channel interaction significantly reduces the Ca(2+) current density in hippocampal neurons. These results underscore the importance of the specific tctex1-channel interaction in determining sorting and trafficking of neuronal Ca(2+) channels with different functionalities.

SummaryTctex1 is a light chain found in both cytoplasmic and flagellar dyneins and is involved in many fundamental cellular activities, including rhodopsin transport within photoreceptors, and may function in the non-Mendelian transmission of t haplotypes in mice. Here, we present the NMR solution structure for the Tctex1 dimer from Chlamydomonas axonemal inner dynein arm I1. Structural comparisons reveal a strong similarity with the LC8 dynein light chain dimer, including formation of a strand-switched β sheet interface. Analysis of the Tctex1 structure enables the dynein intermediate chain binding site to be identified and suggests a mechanism by which cargo proteins might be attached to this microtubule motor complex. Comparison with the alternate dynein light chain rp3 reveals how the specificity of dynein-cargo interactions mediated by these dynein components is achieved. In addition, this structure provides insight into the consequences of the mutations found in the t haplotype forms of this protein.

Chaperonins are key components of the cellular chaperone machinery. These large, cylindrical complexes contain a central cavity that binds to unfolded polypeptides and sequesters them from the cellular environment. Substrate folding then occurs in this central cavity in an ATP-dependent manner. The eukaryotic chaperonin TCP-1 ring complex (TRiC, also called CCT) is indispensable for cell survival because the folding of an essential subset of cytosolic proteins requires TRiC, and this function cannot be substituted by other chaperones. This specificity indicates that TRiC has evolved structural and mechanistic features that distinguish it from other chaperones. Although knowledge of this unique complex is in its infancy, we review recent advances that open the way to understanding the secrets of its folding chamber.

A human gene T-complex protein 10 like (TCP10L) was cloned in our lab. A previous study showed that it expressed specifically in the liver and testis. A transcription experiment revealed that TCP10L was a transcription factor with transcription inhibition activity. In this study, the human MAD4 was identified to interact with TCP10L by a yeast two-hybrid screen. This finding was confirmed by immunoprecipitation and subcellular localization experiments. As MAD4 is a member of the MAD family, which antagonizes the functions of MYC and promotes cell differentiation, the biological function of the interaction between TCP10L and MAD4 may be to maintain the differentiation state in liver cells. Also, we propose that the up-regulation of Myc is caused by the down-regulation of TCP10L in human hepatocarcinomas.

Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. There is increasing evidence that the retrograde transport of herpes simplex virus type 1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Here we report that the herpes simplex virus outer capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and is sufficient to mediate retrograde transport of viral capsids in a cellular model. A library of herpes simplex virus capsid and tegument structural genes was constructed and tested for interactions with dynein subunits in a yeast two-hybrid system. A strong interaction was detected between VP26 and the homologous 14-kDa dynein light chains RP3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to RP3, Tctex1, and intact cytoplasmic dynein complexes. Recombinant herpes simplex virus capsids were constructed either with or without VP26. In pull-down assays VP26+ capsids bound to RP3; VP26-capsids did not. To investigate intracellular transport, the recombinant viral capsids were microinjected into living cells and incubated at 37 degrees C. After 1 h VP26+ capsids were observed to co-localize with RP3, Tctex1, and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, whereas VP26-capsids remained in a random distribution. We propose that VP26 mediates binding of incoming herpes simplex virus capsids to cytoplasmic dynein during cellular infection, through interactions with dynein light chains.

The eukaryotic chaperonin TRiC/CCT mediates folding of an essential subset of newly synthesized proteins, including the tumor suppressor VHL. Here we show that chaperonin binding is specified by two short hydrophobic beta strands in VHL that, upon folding, become buried within the native structure. These TRiC binding determinants are disrupted by tumor-causing point mutations that interfere with chaperonin association and lead to misfolding. Strikingly, while unable to fold correctly in vivo, some of these VHL mutants can reach the native state when refolded in a chaperonin-independent manner. The specificity of TRiC/CCT for extended hydrophobic beta strands may help explain its role in folding aggregation-prone polypeptides. Our findings reveal a class of disease-causing mutations that inactivate protein function by disrupting chaperone-mediated folding in vivo.

Transmission ratio distortion is a dramatic example of non-Mendelian transmission. In mice, t-haplotype males produce dysfunctional +-sperm and normal t-sperm, leading to transmission in favor of t-sperm. Genetic studies have indicated that the t-complex responder locus, Tcr, rescues t-sperm but not +-sperm from defective products of t-complex distorter loci, Tcds. Light chain 1 (LC1) and LC3 from sea urchin sperm outer arm dynein have sequence similarities to Tctex2 and Tctex1, respectively, both of which are wild-type products of Tcds. We show here that LC1 and LC3 are able to make a 1:1 complex. Since Tcr is a member of the Smok (sperm motility kinase) family and LC1 is phosphorylated at the activation of sperm motility in a cAMP-dependent manner, this complex in a dynein motor molecule might be a direct target of Smok/Tcr kinase in a signal cascade that regulates sperm motility. Thus, we designate it as Smoac (sperm motility activating complex).

In the present study we characterized two labor-induced genes, DSCR1 (Down syndrome candidate region 1) and TCTE1L (murine t-complex like), which were identified by suppression subtractive hybridization in the pregnant ovine myometrium. DSCR1 and TCTE1L cDNA sequences were retrieved from a custom-made labor-myometrial cDNA library by hybridization screening. The characterized cDNA sequences include 5'-untranslated region (UTR), coding region and 3'-UTR, which are 12 bp, 351 bp and 1716 bp for TCTE1L, and 64 bp, 594 bp and 1539 bp for DSCR1 respectively. The two cDNA sequences encode proteins of 116 and 197 amino acids for TCTE1L and DSCR1 respectively. Northern analysis further confirmed the significant increases of myometrial DSCR1 and TCTE1L mRNA associated with spontaneous term labor (n=6) compared with gestation-matched controls not in labor (n=6). The abundance of DSCR1 and TCTE1L mRNA was attenuated when myometrial contraction was inhibited by Nimesulide (n=6), a specific prostaglandin H synthase 2 inhibitor. Fetal occupancy greatly upregulated DSCR1 and TCTE1L mRNA in the gravid horn during betamethasone-induced premature labor (n=6) compared with the non-gravid horn not in labor (n=3). Estradiol upregulated TCTE1L mRNA, but had no effect on DSCR1 mRNA expression in the non-pregnant sheep myometrium. Progesterone alone had no effect on both DSCR1 and TCTE1L mRNA expression, however progesterone antagonized estradiol's stimulating effect on myometrial TCTE1L mRNA expression in ovariectomized non-pregnant sheep. Upregulation of DSCR1 and TCTE1L in both betamethasone-induced premature labor and spontaneous term labor and inhibition of their expression by Nimesulide suggest a functional role of these two genes in myometrial activation associated with onset of labor. Mechanical stretch, labor and steroids differentially regulated DSCR1 and TCTE1L mRNA in the pregnant and non-pregnant sheep myometrium.

Casein kinase 2 (CK2) is a ubiquitous, multifunctional eukaryotic serine/threonine kinase that phosphorylates an array of proteins. CK2 is a heterotetramer composed of two catalytic (alpha,alpha(')) and two regulatory (beta) subunits. CK2 plays an essential role in regulatory pathways in cell transformation and proliferation. But the role and function of the individual subunits of CK2, which are not in the holoenzyme, are not yet clear. Northern blot analysis reveals the highest CK2beta activity in mouse testicles and brain. By employing a yeast two-hybrid screen to identify the proteins that interact with CK2beta, we have isolated a cDNA clone encoding a 14-kDa protein with homology to dynein light chains and have designated it as Tctex4. CK2beta interacts specifically with Tctex4 both in a yeast two-hybrid system and in an in vitro interaction assay. Northern blot and in situ hybridization showed that Tctex4 is a novel gene that is expressed in mouse testis.

Chaperonins use ATPase cycling to promote conformational changes leading to protein folding. The prokaryotic chaperonin GroEL requires a cofactor, GroES, which serves as a “lid” enclosing substrates in the central cavity and confers an asymmetry on GroEL required for cooperative transitions driving the reaction. The eukaryotic chaperonin TRiC/CCT does not have such a cofactor but appears to have a “built-in” lid. Whether this seemingly symmetric chaperonin also operates through an asymmetric cycle is unclear. We show that unlike GroEL, TRiC does not close its lid upon nucleotide binding, but instead responds to the trigonal-bipyramidal transition state of ATP hydrolysis. Further, nucleotide analogs inducing this transition state confer an asymmetric conformation on TRiC. Similar to GroEL, lid closure in TRiC confines the substrates in the cavity and is essential for folding. Understanding the distinct mechanisms governing eukaryotic and bacterial chaperonin function may reveal how TRiC has evolved to fold specific eukaryotic proteins.

The voltage-dependent anion-selective channel 1 (VDAC1), i.e. eukaryotic porin, functions as a channel in membranous structures as described for the outer mitochondrial membrane, the cell membrane, endosomes, caveolae, the sarcoplasmatic reticulum, synaptosomes, and post-synaptic density fraction. The identification of VDAC1 interacting proteins may be a promising approach for better understanding the biological context and function of the channel protein. In this study human VDAC1 was used as a bait protein in a two-hybrid screening, which is based on the Sos recruitment system (SRS). hVDAC1 interacts with the dynein light chain Tctex-1 and the heat-shock protein peptide-binding protein 74 (PBP74)/mitochondrial heat-shock protein 70 (mtHSP70)/glucose-regulated protein 75 (GRP75)/mortalin in vivo. Both interactions were confirmed by overlay-assays using recombinant partner proteins and purified hVDAC1. Indirect immunofluorescence on HeLa cells indicates a co-localisation of hVDAC1 with the dynein light chain and the PBP74. In addition, HeLa cells were transfected transiently with enhanced green fluorescent protein (EGFP)–hVDAC1 fusion proteins, which also clearly co-localise with both proteins. The functional relevance of the identified protein interactions was analysed in planar lipid bilayer (PLB) experiments. In these experiments both recombinant binding partners altered the electrophysiological properties of hVDAC1. While rTctex-1 increases the voltage-dependence of hVDAC1 slightly, the rPBP74 drastically minimises the voltage-dependence, indicating a modulation of channel properties in each case. Since the identified proteins are known to be involved in the transport or processing of proteins, the results of this study represent additional evidence of membrane-associated trafficking of the voltage-dependent anion-selective channel 1.

Cytoplasmic dynein is the major minus-end directed microtubule-based motor in eukaryotic cells. It is composed of a number of different subunits including three light chain families: Tctex1, LC8, and roadblock. The incorporation of the roadblock light chains into the cytoplasmic dynein complex had not been determined. There are two roadblock genes in mammals, ROBL-1 and ROBL-2. We find that both members of the roadblock family bind directly to all of the intermediate chain isoforms of mammalian cytoplasmic dynein. This was determined with three complementary approaches. A yeast two-hybrid assay demonstrated that both roadblock light chains interact with intermediate chain isoforms from the IC74-1 and IC74-2 genes in vivo. This was confirmed in vitro with both a solid phase blot overlay assay and a solution-binding assay. The roadblock-binding domain on the intermediate chain was mapped to an approximately 72 residue region. The binding domain is downstream of each of the two alternative splice sites in the intermediate chains. This location is consistent with the finding that both roadblock-1 and roadblock-2 show no binding specificity for a single IC74-1 or IC74-2 intermediate chain isoform. In addition, this roadblock-binding domain is significantly downstream from both the Tctex1- and LC8-binding sites, supporting the hypothesis that multiple light chain family members can bind to the same intermediate chain.

Proper levels of gene expression are important for normal mammalian development. Typically, altered gene dosage caused by karyotypic abnormalities results in embryonic lethality or birth defects. Segmental aneuploidy can be compatible with life but often results in contiguous gene syndromes. The ability to manipulate the mouse genome allows the systematic exploration of regions that are affected by alterations in gene dosage. To explore the effects of segmental haploidy in the mouse t complex on chromosome 17, radiation-induced deletion complexes centered at the Sod2 and D17Leh94 loci were generated in embryonic stem (ES) cells. A small interval was identified that, when hemizygous, caused specific embryonic lethal phenotypes (exencephaly and edema) in most fetuses. The penetrance of these phenotypes was background dependent. Additionally, evidence for parent-of-origin effects was observed. This genetic approach should be useful for identifying genes that are imprinted or whose dosage is critical for normal embryonic development.

The deviation of alleles and chromosomes from Mendelian inheritance is characteristic of the meiotic drive. This review describes the mechanism in question using the best-studied example of transmitted ratio distortion in the heterozygous male mice carrying t-haplotypes. The t-complex is best model for studying the meiotic drive under laboratory conditions. Putative mechanisms of meiotic drive that influence the frequency of t-haplotypes in natural populations are considered, of which prezygotic selection is the most important. The role of meiotic drive in male hybrid sterility is emphasized. The factors and models that determine the phenomenon of meiotic drive are discussed in detail.

Point mutations in a hinge linking the small and large domains of beta-actin result in trapped folding intermediates bound to cytosolic chaperonin CCT. The 30-Angstrom cryo-EM-derived structure of apo-CCT-alpha - actin shows actin opened up across its nucleotide-binding cleft and binding to either of two CCT subunit pairs, CCT beta -CCT delta or CCT epsilon -CCT delta, in a similar 1:4 arrangement. The two main duplicated domains of native actin are linked twice, topologically, by the connecting residues, Q137-S145 and P333-S338, and are tightly held together by hydrogen bonding with bound adenine nucleotide. We carried out a mutational screen to find residues in actin that might be involved in the huge rotations observed in the CCT-bound folding intermediate. When two evolutionarily highly conserved glycine residues of beta -actin, G146 and G150, were changed to proline, both mutant actin proteins were poorly processed by CCT in in vitro translation assays; they become arrested on CCT. A three- dimensional reconstruction of the substrate-bound ring of the apo-CCT-beta actin complex shows that beta -actin G150P is not able to bind across the chaperonin cavity to interact with the CCT delta subunit. beta -actin G150P seems tightly packed and apparently bound only to the CCT beta and CCT epsilon subunits, which further indicates that these CCT subunits drive the interaction between CCT and actin. Hinge opening seems to be critical for actin folding, and we suggest that residues G146 and G150 are important components of the hinge around which the rigid subdomains, presumably already present in early actin folding intermediates, rotate during CCT-assisted folding.

Cytoplasmic dynein is a multi-subunit protein complex in which each subunit is encoded by a few genes. How these subunit isoforms are assembled and regulated to mediate the diverse functions of cytoplasmic dynein is unknown. We previously have shown that two highly conserved 14 kDa dynein light chains, Tctex-1 and RP3, have different cargo-binding abilities. In this report, coimmunoprecipitation revealed that Tctex-1 and RP3 were present in mutually exclusive dynein complexes of brain. Two specific antibodies were used to examine the localization of these two dynein light chains in adult rat hippocampal formation and cerebral cortex. By light microscopy, Tctex-1 and RP3 immunoreactivities exhibited distinct and almost complementary distribution patterns in both brain regions. In hippocampal formation, Tctex-1 immunoreactivity was most enriched in somata of newly generated granule cells and scant in the mature granule and pyramidal cell somata. In contrast, RP3 immunoreactivity was abundant in pyramidal and granule cell somata. Ultrastructural analysis of the dentate gyrus revealed both dynein light chains were associated with various membranous organelles that often were affiliated with microtubules. In addition, Tctex-1 and RP3 immunoreactivities were preferentially and highly enriched on membranous organelles and/or vesicles of axon terminals and dendritic spines, respectively. These results suggest that dynein complexes with different subunit composition, and possibly function, are expressed differentially in a spatially and temporally regulated manner. Furthermore, Tctex-1 and RP3 may play important roles in synaptic functions.

Tctex-1 is a light chain of the cytoplasmic and flagellar dyneins and a candidate for one of the distorter products that cause transmission ratio distortion in mice. We report the identification, characterization, and a preliminary mutational analysis of the function of the Drosophila melanogaster dtctex-1 gene, the putative ortholog of the mammalian tctex-1 gene family. Four P-transposon insertions which disrupt the 5' untranslated region of dtctex-1 are viable in homozygous form but cause male sterility due to the production of non-motile sperm. In males homozygous for dtctex-1 mutant alleles the dtctex-1 transcript is undetectable, while in homozygous females transcripts of lower molecular weight are present. By secondary mobilization of P-element insertions several revertants and new mutant alleles carrying deletions in the 5' UTR region of the gene were produced and characterized by PCR and by Northern analysis.

The minus-ended microtubule motor cytoplasmic dynein contains a number of low molecular weight light chains including the 14-kDa Tctex-1. The assembly of Tctex-1 in the dynein complex and its function are largely unknown. Using partially deuterated, (15)N,(13)C-labeled protein samples and transverse relaxation-optimized NMR spectroscopic techniques, the secondary structure and overall topology of Tctex-1 were determined based on the backbone nuclear Overhauser effect pattern and the chemical shift values of the protein. The data showed that Tctex-1 adopts a structure remarkably similar to that of the 8-kDa light chain of the motor complex (DLC8), although the two light chains share no amino acid sequence homology. We further demonstrated that Tctex-1 binds directly to the intermediate chain (DIC) of dynein. The Tctex-1 binding site on DIC was mapped to a 19-residue fragment immediately following the second alternative splicing site of DIC. Titration of Tctex-1 with a peptide derived from DIC, which contains a consensus sequence R/KR/KXXR/K found in various Tctex-1 target proteins, indicated that Tctex-1 binds to its targets in a manner similar to that of DLC8. The experimental results presented in this study suggest that Tctex-1 is likely to be a specific cargo adaptor for the dynein motor complex.

The Tctex1/Tctex2 family of dynein light chains associates with the intermediate chains at the base of the soluble dynein particle. These components are essential for dynein assembly and participate in specific motor-cargo interactions. To further address the role of these light chains in dynein activity, the structural and biochemical properties of several members of this polypeptide class were examined. Gel filtration chromatography and native gel electrophoresis indicate that recombinantChlamydomonas flagellar Tctex1 exists as a dimer in solution. Furthermore, yeast two-hybrid analysis suggests that this association also occurs in vivo. In contrast, both murine and Chlamydomonas Tctex2 are monomeric. To investigate protein-protein interactions involving these light chains, outer arm dynein from Chlamydomonas flagella was cross-linked using dimethylpimelimidate. Immunoblot analysis of the resulting products revealed the interaction of LC2 (Tctex2) with LC6, which is closely related to the highly conserved LC8 protein found in many enzyme systems, including dynein. Northern dot blot analysis demonstrated that Tctex1/Tctex2 family light chains are differentially expressed both in a tissue-specific and developmentally regulated manner in humans. These data provide further support for the existence of functionally distinct populations of cytoplasmic dynein with differing light chain content.

The carboxy terminus-encoding portion of the gag gene of Mason-Pfizer monkey virus (M-PMV), the prototype immunosuppressive primate type D retrovirus, encodes a 36-amino-acid, proline-rich protein domain that, in the mature virion, becomes the p4 capsid protein. The p4 domain has no known role in M-PMV replication. We found that two mutants with premature termination codons that remove half or all of the p4 domain produced lower levels of stable Gag protein and of self-assembled capsids. Interestingly, yeast two-hybrid screening revealed that p4 specifically interacted with TCP-1γ, a subunit of the chaperonin TRiC (TCP-1 ring complex). TRiC is a cytosolic chaperonin that is known to be involved in both folding and subunit assembly of a variety of cellular proteins. TCP-1γ also associated with high specificity with the M-PMV pp24/16-p12 domain and human immunodeficiency virus p6. Moreover, in cells, Gag polyprotein associated with the TRiC chaperonin complex and this association depended on ATP hydrolysis. In the p4 truncation mutants, the Gag-TRiC association was significantly reduced. These results strongly suggest that cytosolic chaperonin TRiC is involved in Gag folding and/or capsid assembly. We propose that TRiC associates transiently with nascent M-PMV Gag molecules to assist in their folding. Consequently, properly folded Gag molecules carry out the intermolecular interactions involved in self-assembly of the immature capsid.

The higher plant, Arabidopsis thaliana, has a large number of genes, each of which encodes a component of His-to-Asp phosphorelay signal transduction systems. One type of such signal transducers are the histidine-containing phosphotransmitters (termed AHPs), which presumably mediate His-to-Asp phosphorelay. Here we attempted to isolate a factor or factors that interact with AHP1, AHP2 and AHP3 by means of a yeast two-hybrid system. This allowed us to identify two types of nuclear-localizing proteins. They are the members of the type-B family of response regulators (specifically, ARR1, APP2 and ARR10), and a novel protein named TCP10. The binding of ARR1 to AHP2 was also confirmed by in vitro binding assays. Moreover, dephosphorylation of AHP2 was observed in a manner dependent on ARR in vitro. A subset of AHPs appeared to also interact with a protein that contains a TCP domain, a recently proposed basic helix-loop-helix motif. Because several factors carrying the TCP domain have been implicated in the regulation of growth and development in lateral organs, the binding of TCP10 to this subset of AHPs suggests a possible linkage between the His-to-Asp phosphorelay systems and plant growth regulation.

The t haplotypes (t) are recent evolutionary derivatives of an alternate form of the mouse t complex region located at the proximal end of chromosome 17. This variant form of approximately 1% of the mouse genome is a source of mutations altering numerous sperm functions crucial for fertilization. Males that carry two t haplotypes (t/t) are invariably sterile. t haplotypes contain four inversions relative to the wild-type t complex (+), so that in matings involving a +/t heterozygote, t is usually transmitted as a single unit. However, rare recombinants have been recovered, which carry only part of the t genotype and express only some of the t-dependent phenotypes. Use of these partial t haplotypes in genetic crosses has resulted in the general location of the two major t male sterility factors, S1 and S2, within inversions 1 and 4, respectively. Since sterility can result from a plethora of sperm defects, we have made a detailed study of various functional parameters of sperm from mice carrying S1 or S2 heterozygously or homozygously or in combination. Both S1 and S2 contain mutations altering sperm functions, including motility, capacitation, binding to the zona pellucida, binding to the oocyte membrane, and penetration of the zona pellucida-free oocyte. Therefore it seems clear that each of these factors contains multiple genes contributing to sterility. Furthermore, our results indicate that genes within S1 interact with genes in S2 for all sperm functions examined. However, S1 and S2 genes affecting motility interact in a purely additive fashion, while S1 and S2 genes affecting most other sperm characteristics interact in a synergistic manner. Additionally, the patterns of synergism between S1 and S2 for abnormalities in capacitation, sperm–oolemma binding, and zona-free oocyte penetration are nearly identical. This suggests that these three defects are caused by mutation of the same gene within each sterility factor. These findings will not only be instrumental in matching the various t haplotype sperm defects to candidate genes for S1 and S2, but will facilitate a more comprehensive understanding of the cellular and genetic mechanisms underlying t haplotype male sterility.

The solvent ethylene glycol monomethyl ether (EGME) produces the same testicular lesions in rodents and human testis cultures, whose onset is characterized by apoptosis of pachytene spermatocytes. To identify gene changes early in the lesion and determine the possible involvement of cells other than the spermatocytes, we employed a suppression subtractive hybridization technique using whole testes from mice treated 8 h previously with 500 mg/kg EGME to generate two subtracted mouse testis cDNA libraries enriched for gene populations either up-regulated or down-regulated by EGME. A total of 70 clones were screened, and 6 of them were shown by Northern blotting to be differentially expressed in the EGME lesion. The three clones with increased expression after EGME treatment were identical to t-complex testis expressed gene 1 (tctex1), a gene encoding ribosomal protein S25, and a heretofore uncharacterized mouse testis expressed sequence tag. Three other genes suppressed by EGME were tctex2, alpha-2,6-sialyltransferase gene, and another uncharacterized mouse testis expressed sequence tag. Predicted peptide sequences of these clones contain multiple motifs for phosphorylation, glycosylation, and myristoylation. In situ hybridization with the antisense RNA probes further supported the expression changes of these six clones and localized the changes in multiple germ cell stages as well as other cell types (Sertoli, interstitial and peritubular cells). These data at the gene expression level are the first to demonstrate the early involvement in this lesion of cell types other than the dying spermatocytes.