Pseudovacuoles--immobilized by high-pressure freezing--are associated with blebbing in walker carcinosarcoma cells

By applying high pressure freezing and freeze-substitution, we observed large inclusions of homogeneous appearance in the front of locomoting Walker carcinosarcoma cells that have not been described earlier. Live cell imaging revealed that these inclusions were poor in lipids and nucleic acids but had a high lysine (and hence protein) content. Usually one such structure 2-5 mum in size was present at the front of motile Walker cells, predominantly in the immediate vicinity of newly forming blebs. By correlating the lysine-rich areas in fixed and embedded cells with electron microscopic pictures, inclusions could be assigned to confined, faintly stained cytoplasmic areas that lacked a surrounding membrane; they were therefore called pseudovacuoles. After high-pressure freezing and freeze substitution, pseudovacuoles appeared to be filled with 20 nm large electron-transparent patches surrounded by 12 and 15 nm large particles. The heat shock protein Hsp90 was identified by peptide sequencing as a major fluorescent band on SDS-PAGE of lysine-labelled Walker cell extracts. By immunofluorescence, Hsp90 was found to be enriched in pseudovacuoles. Colocalization of the lysine with a potassium-specific dye in living cells revealed that pseudovacuoles act as K+ stores in the vicinity of forming blebs. We propose that pseudovacuoles might support blebbing by locally regulating the intracellular hydrostatic pressure.

A Deletion in the VLDLR Gene in Eurasier Dogs with Cerebellar Hypoplasia Resembling a Dandy-Walker-Like Malformation (DWLM).

Dandy-Walker-like malformation (DWLM) is the result of aberrant brain development and mainly characterized by cerebellar hypoplasia. DWLM affected dogs display a non-progressive cerebellar ataxia. Several DWLM cases were recently observed in the Eurasier dog breed, which strongly suggested a monogenic autosomal recessive inheritance in this breed. We performed a genome-wide association study (GWAS) with 9 cases and 11 controls and found the best association of DWLM with markers on chromosome 1. Subsequent homozygosity mapping confirmed that all 9 cases were homozygous for a shared haplotype in this region, which delineated a critical interval of 3.35 Mb. We sequenced the genome of an affected Eurasier and compared it with the Boxer reference genome and 47 control genomes of dogs from other breeds. This analysis revealed 4 private non-synonymous variants in the critical interval of the affected Eurasier. We genotyped these variants in additional dogs and found perfect association for only one of these variants, a single base deletion in the VLDLR gene encoding the very low density lipoprotein receptor. This variant, VLDLR:c.1713delC is predicted to cause a frameshift and premature stop codon (p.W572Gfs*10). Variants in the VLDLR gene have been shown to cause congenital cerebellar ataxia and mental retardation in human patients and Vldlr knockout mice also display an ataxia phenotype. Our combined genetic data together with the functional knowledge on the VLDLR gene from other species thus strongly suggest that VLDLR:c.1713delC is indeed causing DWLM in Eurasier dogs.

Robert Walker [Illustration] : (Porträt)

Samuel Cooper

Sarah Jane Walker, Kniestück

Signatur des Originals: S 36/F03308

Sarah Jane Walker, Hüftbild

Signatur des Originals: S 36/F03309

Sarah Jane Walker, Rollenbild, Brustbild

Signatur des Originals: S 36/F03310

Edyth Walker, Brustbild

Signatur des Originals: S 36/F07137

Edyth Walker, Brustbild

Signatur des Originals: S 36/F07138

Edyth Walker als Isolde, Rollenbild, Kniestück, in: Richard Wagner: Tristan und Isolde

Signatur des Originals: S 36/F07139

Edyth Walker als Elektra, Szenenfoto, in: Richard Strauss: Elektra

Signatur des Originals: S 36/F07140

Edyth Walker als Elektra, Szenenfoto, in: Richard Strauss: Elektra

Signatur des Originals: S 36/F07141

Edyth Walker, Rollenbild, Brustbild

Signatur des Originals: S 36/F10190

Edyth Walker, Rollenbild, Kniestück

Signatur des Originals: S 36/F10191

Edyth Walker als Fidelio, Rollenbild, in: Ludwig van Beethoven: Fidelio

Signatur des Originals: S 36/F11263