Genetic heterogeneity in Pakistani microcephaly families revisited.

Autosomal recessive primary microcephaly (MCPH) is a rare and heterogeneous genetic disorder characterized by reduced head circumference, low cognitive prowess and, in general, architecturally normal brains. As many as 14 different loci have already been mapped. We recruited 35 MCPH families in Pakistan and could identify the genetic cause of the disease in 31 of them. Using homozygosity mapping complemented with whole-exome, gene panel or Sanger sequencing, we identified 12 novel mutations in 3 known MCPH-associated genes - 9 in ASPM, 2 in MCPH1 and 1 in CDK5RAP2. The 2 MCPH1 mutations were homozygous microdeletions of 164,250 and 577,594 bp, respectively, for which we were able to map the exact breakpoints. We also identified four known mutations - three in ASPM and one in WDR62. The latter was initially deemed to be a missense mutation but we demonstrate here that it affects splicing. As to ASPM, as many as 17 out of 27 MCPH5 families that we ascertained in our sample were found to carry the previously reported founder mutation p.Trp1326*. This study adds to the mutational spectra of four known MCPH-associated genes and updates our knowledge about the genetic heterogeneity of MCPH in the Pakistani population considering its ethnic diversity.

Dictyostelium genomics: how it developed and what we have learned from it.

Dictyostelium discoideum is the most prominent member of the social amoebae. It has been used as an experimental system since more than 50 years and a large number of scientists worldwide work on different aspects such as chemotaxis, cytoskeleton, differentiation and development. Dictyostelium shares more features with animals than fungi although it diverged much earlier in evolution. Many of the results obtained with D. discoideum can therefore be transferred to animals making D. discoideum a valuable model organism. Targeted gene inactivation using homologous recombination is easy and mutant phenotypes can be readily isolated due to the haploid nature of its genome. Furthermore, a variety of techniques and tools are available that facilitate the experimental work; its genome and that of several Dictyostelidae has been sequenced and most recently a high-resolution genome wide nucleosome map for D. discoideum has been generated.

Genetic heterogeneity in Pakistani microcephaly families.

Autosomal recessive primary microcephaly (MCPH) is caused by mutations in at least eight different genes involved either in cell division or DNA repair. Most mutations are identified in consanguine families from Pakistan, Iran and India. To further assess their genetic heterogeneity and mutational spectra, we have analyzed 57 consanguine Pakistani MCPH families. In 34 MCPH families, we detected linkage to five out of the eight well-characterized disease loci and identified mutations in 27 families, leaving seven families without mutations in the coding exons of the presumably underlying MCPH genes. In the MCPH cohort 23 families could not be linked to any of the known loci, pointing to remarkable locus heterogeneity. The majority of mutations were found in ASPM followed by WDR62, CENPJ, CEP152 and MCPH1. One ASPM mutation (p.Trp1326*) was found in as many as eight families suggesting a Pakistani founder mutation. One third of the families were linked to ASPM followed by WDR62 confirming previous data. We identified three novel ASPM mutations, four novel WDR62 mutations, one novel MCPH1 mutation and two novel CEP152 mutations. CEP152 mutations have not been described before in the Pakistani population.

Molecular mechanism underlying the association of Coronin-7 with Golgi membranes.

Coronin-7 (Crn7) is a ubiquitous mammalian WD40-repeat protein that localizes to the Golgi complex, interacts with AP-1 adaptor complex via binding of a tyrosine-288-based sorting signal to the mu1-subunit of AP-1, and participates in the maintenance of the Golgi structure and function. Here, we define the requirements for the recruitment of Crn7 from the cytosol to the Golgi. We establish that Src activity is indispensable for the interaction of Crn7 with Golgi membranes. Crn7 binds Src in vivo and can be phosphorylated by recombinant Src in vitro. We demonstrate that tyrosine-758 is the major Src phosphorylation site. Further, to be targeted to membranes Crn7 requires the presence of cargo in the Golgi complex. Finally, downregulation of the mu1-subunit of AP-1 leads to the dispersal of Crn7 from the Golgi membranes. We propose a mechanism whereby sequential events of protein interaction and posttranslational modification result in the membrane targeting of Crn7.

CAP2, cyclase-associated protein 2, is a dual compartment protein.

Cyclase-associated proteins (CAPs) are evolutionarily conserved proteins with roles in regulating the actin cytoskeleton and in signal transduction. Mammals have two CAP genes encoding the related CAP1 and CAP2. We studied the distribution and subcellular localization of CAP1 and CAP2 using specific antibodies. CAP1 shows a broad tissue distribution, whereas CAP2 is significantly expressed only in brain, heart and skeletal muscle, and skin. CAP2 is found in the nucleus in undifferentiated myoblasts and at the M-line of differentiated myotubes. In PAM212, a mouse keratinocyte cell line, CAP2 is enriched in the nucleus, and sparse in the cytosol. By contrast, CAP1 localizes to the cytoplasm in PAM212 cells. In human skin, CAP2 is present in all living layers of the epidermis localizing to the nuclei and the cell periphery. In in vitro studies, a C-terminal fragment of CAP2 interacts with actin, indicating that CAP2 has the capacity to bind to actin.

LIM proteins: association with the actin cytoskeleton.

The LIM domain is an evolutionary conserved double-zinc finger motif found in a variety of proteins exhibiting diverse biological roles. LIM domains have been observed to act as modular protein-binding interfaces mediating protein-protein interactions in the cytoplasm and the nucleus. Interaction of LIM domains with specific protein partners is now known to influence its subcellular localization and activity; however, no single binding motif has been identified as a common target for LIM domains. Several LIM domain-containing proteins associated with the actin cytoskeleton have been identified, playing a role in signal transduction and organization of the actin filaments during various cellular processes.

Villin-type headpiece domains show a wide range of F-actin-binding affinities.

The villin-type "headpiece" domain is a modular motif found at the extreme C-terminus of larger "core" domains in over 25 cytoskeletal proteins in plants and animals. Although headpiece is classified as an F-actin-binding domain, it has been suggested that some expressed fusion-proteins containing headpiece may lack F-actin-binding in vivo. To determine the intrinsic F-actin affinity of headpiece domains, we quantified the F-actin affinity of seven headpiece domains and three N-terminal truncations, under identical in vitro conditions. The constructs are folded and adopt the native headpiece structure. However, they show a wide range of affinities that can be grouped into high, low, and nonspecific-binding categories. Computer models of the structure and charged surface potential of these headpiece domains suggest features important for high F-actin affinity. We conclude that not all headpiece domains are intrinsically F-actin-binding motifs, and suggest that the surface charge distribution may be an important element for F-actin recognition.

Genomic organization and expression profile of the parvin family of focal adhesion proteins in mice and humans.

We have characterized the genomic organization and the expression pattern of alpha-, beta- and gamma-parvin, a novel family of focal adhesion proteins, in mice and humans. alpha-Parvin is nearly ubiquitously expressed, beta-parvin is preferentially expressed in heart- and skeletal muscle, and gamma-parvin in lymphoid tissues. Parvins display diverse patterns of developmental regulation. The alpha-form is present throughout mouse development, beta-parvin is gradually upregulated and gamma-parvin is downregulated at embryonic day 11. The human alpha-parvin gene (PARVA), extending over 160 kb, is located on chromosome 11. Both, the human beta-parvin gene (PARVB), which is over 145 kb long, and the gamma-parvin gene (PARVG) of a total length of about 25 kb are positioned on chromosome 22 with PARVG located about 12 kb downstream of the 3' end of PARVB. Multiple tissue array analysis indicates that parvins are expressed at reduced levels in cancer as compared to the corresponding normal tissues. Analysis of ESTs and PCR-amplified fragments reveals alternatively spliced and alternatively polyadenylated gene products. Mammalian parvins are likely to have arisen late in evolution from gene duplication as they share a remarkably similar exon/intron organization, which is different from the organization of the single genes encoding parvin-like proteins in Drosophila and Caenorhabditis.

Loss of annexin A7 leads to alterations in frequency-induced shortening of isolated murine cardiomyocytes.

Annexin A7 has been proposed to function in the fusion of vesicles, acting as a Ca(2+) channel and as Ca(2+)-activated GTPase, thus inducing Ca(2+)/GTP-dependent secretory events. To understand the function of annexin A7, we have performed targeted disruption of the Anxa7 gene in mice. Matings between heterozygous mice produced offspring showing a normal Mendelian pattern of inheritance, indicating that the loss of annexin A7 did not interfere with viability in utero. Mice lacking annexin A7 showed no obvious phenotype and were fertile. To assay for exocytosis, insulin secretion from isolated islets of Langerhans was examined. Ca(2+)-induced and cyclic AMP-mediated potentiation of insulin secretion was unchanged in the absence of annexin A7, suggesting that it is not directly implicated in vesicle fusion. Ca(2+) regulation studied in isolated cardiomyocytes, showed that while cells from early embryos displayed intact Ca(2+) homeostasis and expressed all of the components required for excitation-contraction coupling, cardiomyocytes from adult Anxa7(-/-) mice exhibited an altered cell shortening-frequency relationship when stimulated with high frequencies. This suggests a function for annexin A7 in electromechanical coupling, probably through Ca(2+) homoeostasis.

Annexin VII: an astroglial protein exhibiting a Ca2+-dependent subcellular distribution.

A fundamental issue in neuronal and glial cells is how intracellular rises in Ca2+ are coupled to signaling cascades and changes in subcellular morphology. We studied the expression and localization of annexin VII (synexin), a Ca(2+)-/GTP-dependent membrane fusion protein, in the human CNS. Here, we demonstrate the presence of two annexin VII isoforms (47 and 51 kDa) in human brain tissue as well as its exclusive expression in astroglial cells. An in vitro study of astrocyte-derived C6 rat glioblastoma cells expressing a GFP tagged annexin VII fusion protein demonstrates a sequential redistribution of the fusion protein in response to rising intracellular Ca2+ concentrations. Our findings indicate a role of annexin VII in the regulation of intracellular Ca(2+)-dependent processes in astroglial cells.

The complex repeats of Dictyostelium discoideum.

In the course of determining the sequence of the Dictyostelium discoideum genome we have characterized in detail the quantity and nature of interspersed repetitive elements present in this species. Several of the most abundant small complex repeats and transposons (DIRS-1; TRE3-A,B; TRE5-A; skipper; Tdd-4; H3R) have been described previously. In our analysis we have identified additional elements. Thus, we can now present a complete list of complex repetitive elements in D. discoideum. All elements add up to 10% of the genome. Some of the newly described elements belong to established classes (TRE3-C, D; TRE5-B,C; DGLT-A,P; Tdd-5). However, we have also defined two new classes of DNA transposable elements (DDT and thug) that have not been described thus far. Based on the nucleotide amount, we calculated the least copy number in each family. These vary between <10 up to >200 copies. Unique sequences adjacent to the element ends and truncation points in elements gave a measure for the fragmentation of the elements. Furthermore, we describe the diversity of single elements with regard to polymorphisms and conserved structures. All elements show insertion preference into loci in which other elements of the same family reside. The analysis of the complex repeats is a valuable data resource for the ongoing assembly of whole D. discoideum chromosomes.

Parvin, a 42 kDa focal adhesion protein, related to the alpha-actinin superfamily.

We have identified and cloned a novel 42-kDa protein termed alpha-parvin, which has a single alpha-actinin-like actin-binding domain. Unlike other members of the alpha-actinin superfamily, which are large multidomain proteins, alpha-parvin lacks a rod domain or any other C-terminal structural modules and therefore represents the smallest known protein of the superfamily. We demonstrate that mouse alpha-parvin is widely expressed as two mRNA species generated by alternative use of two polyadenylation signals. We analyzed the actin-binding properties of mouse alpha-parvin and determined the K(d) with muscle F-actin to be 8.4+/-2.1 microM. The GFP-tagged alpha-parvin co-localizes with actin filaments at membrane ruffles, focal contacts and tensin-rich fibers in the central area of fibroblasts. Domain analysis identifies the second calponin homology domain of parvin as a module sufficient for targeting the focal contacts. In man and mouse, a closely related paralogue beta-parvin and a more distant relative gamma-parvin have also been identified and cloned. The availability of the genomic sequences of different organisms enabled us to recognize closely related parvin-like proteins in flies and worms, but not in yeast and Dictyostelium. Phylogenetic analysis of alpha-parvin and its para- and orthologues suggests, that the parvins represent a new family of alpha-actinin-related proteins that mediate cell-matrix adhesion.

The Dictyostelium discoideum family of Rho-related proteins.

Taking advantage of the ongoing Dictyostelium genome sequencing project, we have assembled >73 kb of genomic DNA in 15 contigs harbouring 15 genes and one pseudogene of Rho-related proteins. Comparison with EST sequences revealed that every gene is interrupted by at least one and up to four introns. For racC extensive alternative splicing was identified. Northern blot analysis showed that mRNAs for racA, racE, racG, racH and racI were present at all stages of development, whereas racJ and racL were expressed only at late stages. Amino acid sequences have been analysed in the context of Rho-related proteins of other organisms. Rac1a/1b/1c, RacF1/F2 and to a lesser extent RacB and the GTPase domain of RacA can be grouped in the Rac subfamily. None of the additional Dictyostelium Rho-related proteins belongs to any of the well-defined subfamilies, like Rac, Cdc42 or Rho. RacD and RacA are unique in that they lack the prenylation motif characteristic of Rho proteins. RacD possesses a 50 residue C-terminal extension and RacA a 400 residue C-terminal extension that contains a proline-rich region, two BTB domains and a novel C-terminal domain. We have also identified homologues for RacA in Drosophila and mammals, thus defining a new subfamily of Rho proteins, RhoBTB.

Polymerization, three-dimensional structure and mechanical properties of Ddictyostelium versus rabbit muscle actin filaments.

To assess more systematically functional differences among non-muscle and muscle actins and the effect of specific mutations on their function, we compared actin from Dictyostelium discoideum (D-actin) with actin from rabbit skeletal muscle (R-actin) with respect to the formation of filaments, their three-dimensional structure and mechanical properties. With Mg(2+) occupying the single high-affinity divalent cation-binding site, the course of polymerization is very similar for the two types of actin. In contrast, when Ca(2+ )is bound, D-actin exhibits a significantly longer lag phase at the onset of polymerization than R-actin. Crossover spacing and helical screw angle of negatively stained filaments are similar for D and R-F-actin filaments, irrespective of the tightly bound divalent cation. However, three-dimensional helical reconstructions reveal that the intersubunit contacts along the two long-pitch helical strands of D-(Ca)F-actin filaments are more tenuous compared to those in R-(Ca)F-actin filaments. D-(Mg)F-actin filaments on the other hand exhibit more massive contacts between the two long-pitch helical strands than R-(Mg)F-actin filaments. Moreover, in contrast to the structure of R-F-actin filaments which is not significantly modulated by the divalent cation, the intersubunit contacts both along and between the two long-pitch helical strands are weaker in D-(Ca)F-actin compared to D-(Mg)F-actin filaments. Consistent with these structural differences, D-(Ca)F-actin filaments were significantly more flexible than D-(Mg)F-actin. Taken together, this work documents that despite being highly conserved, muscle and non-muscle actins exhibit subtle differences in terms of their polymerization behavior, and the three-dimensional structure and mechanical properties of their F-actin filaments which, in turn, may account for their functional diversity.

Cloning and characterization of beta-COP from Dictyostelium discoideum.

We have isolated a cDNA coding for beta-COP from Dictyostelium discoideum by polymerase chain reaction using degenerate primers derived from rat beta-COP. The complete cDNA clone has a size of 2.8 kb and codes for a protein with a calculated molecular mass of 102 kDa. Dictyostelium beta-COP exhibits highest homology to mammalian beta-COP, but it is considerably smaller due to a shortened variable region that is thought to form a linker between the highly conserved N- and C-terminal domains. Dictyostelium beta-COP is encoded by a single gene, which is transcribed at moderate levels into two RNAs that are present throughout development. To localize the protein, full-length beta-COP was fused to GFP and expressed in Dictyostelium cells. The fusion protein was detected on vesicles distributed all over the cells and was strongly enriched in the perinuclear region. Based on coimmunofluorescence studies with antibodies directed against the Golgi marker comitin, this compartment was identified as the Golgi apparatus. Beta-COP distribution in Dictyostelium was not brefeldin A sensitive being most likely due to the presence of a brefeldin A resistance gene. However, upon DMSO treatment we observed a reversible disassembly of the Golgi apparatus. In mammalian cells DMSO treatment had a similar effect on beta-COP distribution.

The sorcin-annexin VII calcium-dependent interaction requires the sorcin N-terminal domain.

Surface plasmon resonance experiments show that at neutral pH the stability of the complex between sorcin and annexin VII (synexin) increases dramatically between 3 and 6 microM calcium; at the latter cation concentration the K(D) value is 0.63 microM. In turn, the lack of complex formation between the sorcin Ca(2+) binding domain (33-198) and synexin maps the annexin binding site to the N-terminal region of the sorcin polypeptide chain. Annexin VII likewise employs the N-terminal domain, more specifically the first 31 amino acids, to interact with sorcin [Brownawell, A.M. and Creutz, C.E. (1997) J. Biol. Chem. 272, 22182-22190]. The interaction may involve similar structural motifs in the two proteins, namely GGYY and GYGG in sorcin and GYPP in synexin.

Rapid and reproducible quantification of hepatitis C virus cDNA by fluorescence correlation spectroscopy.

BACKGROUND/AIMS: Standard methods for hepatitis C virus (HCV) RNA quantification are time-consuming and often hampered by low sensitivity. Therefore, we aimed to test whether fluorescence correlation spectroscopy (FCS) could be used to read out HCV polymerase chain reactions (PCR). METHODS: A single-step reverse transcriptase (RT) PCR system was adjusted to the clinically relevant range of 1 x 10(3) to 5 x 10(6) HCV cDNA copies/ml serum. Unpurified amplification mixtures were analyzed by FCS and controlled by HPLC analysis. RESULTS: The outcome of HCV RNA quantitation was nearly identical no matter whether FCS or HPLC techniques were used. FCS-generated standard curves displayed sufficient linearity to allow reproducible determinations. The intraserial variation of cDNA quantification after PCR amplification was +/-3.2%, the interserial variation +/-4.3%. Repeated quantifications of HCV genotype 1b RNA from the sera of 8 patients revealed titers from 1 x 10(4)-5 x 10(6) genome equivalents/ml. The results correlated significantly (r = 0.755; p = 0.03) with a widely used commercially available assay. CONCLUSION: FCS may become a useful tool for rapid and reproducible HCV RNA quantification in the future.

Dissection of functional domains by expression of point-mutated profilins in Dictyostelium mutants.

Profilin is a ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cell physiology. By site-directed mutagenesis of profilin II from Dictyostelium discoideum the point mutations K114E and W3N were generated by PCR thus changing actin and poly-(L)-proline-binding activity respectively. W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin binding. The K114E profilin exhibited a profound decrease in its ability to interact with actin, whereas binding to poly-(L)-proline was essentially unchanged. Binding to phospholipids was indistinguishable from the wild-type profilin. The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in profilin-minus D. discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., Cell 79, 303-314, 1994). Expression of K114E or W3N displayed a reduction in the F-actin content, normal cell morphology, and the transformants were capable of undergoing complete development. Interestingly, only cells that drastically overexpressed W3N could restore the aberrant phenotype, whereas the mutant protein K114E with its fully functional poly-(L)-proline binding and its strongly reduced actin-binding activities rescued the phenotype at low concentrations. Wild-type and both mutated profilins are enriched in phagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline-binding activity is more important for suppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities.

The actin cytoskeleton of Dictyostelium: a story told by mutants.

Actin-binding proteins are effectors of cell signalling and coordinators of cellular behaviour. Research on the Dictyostelium actin cytoskeleton has focused both on the elucidation of the function of bona fide actin-binding proteins as well as on proteins involved in signalling to the cytoskeleton. A major part of this work is concerned with the analysis of Dictyostelium mutants. The results derived from these investigations have added to our understanding of the role of the actin cytoskeleton in growth and development. Furthermore, the studies have identified several cellular and developmental stages that are particularly sensitive to an unbalanced cytoskeleton. In addition, use of GFP fusion proteins is revealing the spatial and temporal dynamics of interactions between actin-associated proteins and the cytoskeleton.

Assessing the role of the ASP56/CAP homologue of Dictyostelium discoideum and the requirements for subcellular localization.

The CAP (cyclase-associated protein) homologue of Dictyostelium discoideum is a phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulated G-actin sequestering protein which is present in the cytosol and shows enrichment at plasma membrane regions. It is composed of two domains separated by a proline rich stretch. The sequestering activity has been localized to the C-terminal domain of the protein, whereas the presence of the N-terminal domain seems to be required for PIP(2)-regulation of the sequestering activity. Here we have constructed GFP-fusions of N- and C-domain and found that the N-terminal domain showed CAP-specific enrichment at the anterior and posterior ends of cells like endogenous CAP irrespective of the presence of the proline rich region. Mutant cells expressing strongly reduced levels of CAP were generated by homologous recombination. They had an altered cell morphology with very heterogeneous cell sizes and exhibited a cytokinesis defect. Growth on bacteria was normal both in suspension and on agar plates as was phagocytosis of yeast and bacteria. In suspension in axenic medium mutant cells grew more slowly and did not reach saturation densities observed for wild-type cells. This was paralleled by a reduction in fluid phase endocytosis. Development was delayed by several hours under all conditions assayed, furthermore, motile behaviour was affected.