Lsb1 Is a Negative Regulator of Las17 Dependent Actin Polymerization Involved in Endocytosis

The spatial and temporal regulation of actin polymerization is crucial for various cellular processes. Members of the Wiskott–Aldrich syndrome protein (WASP) family activate the Arp2/3-complex leading to actin polymerization. The yeast Saccharomyces cerevisiae contains only one WASP homolog, Las17, that requires additional factors for its regulation. Lsb1 and Lsb2/Pin3 are two yeast homologous proteins bearing an SH3 domain that were identified as Las17-binding proteins. Lsb2/Pin3 that promotes prion induction was suggested to link this prion formation to the actin cytoskeleton. However, the cellular role of Lsb1 and the molecular function of both Lsb1 and Lsb2 remain unknown. In this study, we show that Lsb1 and/or Lsb2 full-length proteins inhibit Las17-mediated actin polymerization in vitro, Lsb2 being a less potent inhibitor of Las17 activity compared to Lsb1. Addition of Lsb1 or Lsb2 to the corresponding full-length Lsb1/2 further inhibits Las17 activity. Lsb1 and Lsb2 form homo- and hetero-oligomeric complexes suggesting that these two proteins could regulate Las17 activity via dimerization or cooperative binding. In vivo, overexpressed Lsb1 and Lsb2 proteins cluster Las17-CFP in few cytoplasmic punctate structures that are also positive for other Arp2/3-dependent actin polymerization effectors like Sla1 or Abp1. But, only Lsb1 overexpression blocks the internalization step of receptor-mediated endocytosis. This shows a specific function of Lsb1 in endocytosis.     

Ent3p Is a PtdIns(3,5)P2 effector required for protein sorting to the multivesicular body

PtdIns(3,5)P(2) is required for cargo-selective sorting to the vacuolar lumen via the multivesicular body (MVB). Here we show that Ent3p, a yeast epsin N-terminal homology (ENTH) domain-containing protein, is a specific PtdIns(3,5)P(2) effector localized to endosomes. The ENTH domain of Ent3p is essential for its PtdIns(3,5)P(2) binding activity and for its membrane interaction in vitro and in vivo. Ent3p is required for protein sorting into the MVB but not for the internalization step of endocytosis. Ent3p is associated with clathrin and is necessary for normal actin cytoskeleton organization. Our results show that Ent3p is required for protein sorting into intralumenal vesicles of the MVB through PtdIns(3,5)P(2) binding via its ENTH domain.     

Osteoclast cell-surface changes during the egg-laying cycle in Japanese quail

The medullary bone serves as a source of labile calcium mobilized during calcification of the egg shell in birds. Quantitative histological methods demonstrate that the numbers of medullary bone osteoclasts and nuclei per osteoclast remain unchanged during the egg cycle in the Japanese quail (Coturnix). Therefore, cyclic changes in bone resorption cannot be explained by modulations of osteoclasts from and into other bone cells, a mechanism previously suggested for certain species of birds. Rather, dramatic changes in osteoclast cell-surface features occur during the egg cycle, which might account for cyclic variations in resorptive activity. During egg shell calcification, osteoclasts with ruffled borders are closely apposed to bone surfaces; the cytoplasm is rich in vacuoles that contain mineral crystals and seem to derive from the ruffled border. At the completion of egg shell calcification, the ruffled borders and vacuoles move away from the bone surface, although the osteoclast remains attached to the bone along the filamentous or "clear" zone. Associated with the disappearance of the ruffled borders is the appearance of extensive interdigitated cell processes along the peripheral surface of the osteoclast away from the bone. These unusual structures, which may serve as a reservoir of membrane, largely disappear when ruffled borders and associated structures reappear. Therefore, in these hens, the osteoclasts modulate their cell surface rather than their population during the egg cycle.     

Ent5p is required with Ent3p and Vps27p for ubiquitin-dependent protein sorting into the multivesicular body

At the late endosomes, cargoes destined for the interior of the vacuole are sorted into invaginating vesicles of the multivesicular body. Both PtdIns(3,5)P₂ and ubiquitin are necessary for proper sorting of some of these cargoes. We show that Ent5p, a yeast protein of the epsin family homologous to Ent3p, localizes to endosomes and specifically binds to PtdIns(3,5)P₂ via its ENTH domain. In cells lacking Ent3p and Ent5p, ubiquitin-dependent sorting of biosynthetic and endocytic cargo into the multivesicular body is disrupted, whereas other trafficking routes to the vacuole are not affected. Ent3p and Ent5p are associated with Vps27p, a FYVE domain containing protein that interacts with ubiquitinated cargoes and is required for protein sorting into the multivesicular body. Therefore, Ent3p and Ent5p are the first proteins shown to be connectors between PtdIns(3,5)P₂- and the Vps27p-ubiquitin-driven sorting machinery at the multivesicular body.     

Effects of sex chromosome dosage on corpus callosum morphology in supernumerary sex chromosome aneuploidies

Background

Supernumerary sex chromosome aneuploidies (sSCA) are characterized by the presence of one or more additional sex chromosomes in an individual’s karyotype; they affect around 1 in 400 individuals. Although there is high variability, each sSCA subtype has a characteristic set of cognitive and physical phenotypes. Here, we investigated the differences in the morphometry of the human corpus callosum (CC) between sex-matched controls 46,XY (N =99), 46,XX (N =93), and six unique sSCA karyotypes: 47,XYY (N =29), 47,XXY (N =58), 48,XXYY (N =20), 47,XXX (N =30), 48,XXXY (N =5), and 49,XXXXY (N =6).

Methods

We investigated CC morphometry using local and global area, local curvature of the CC boundary, and between-landmark distance analysis (BLDA). We hypothesized that CC morphometry would vary differentially along a proposed spectrum of Y:X chromosome ratio with supernumerary Y karyotypes having the largest CC areas and supernumerary X karyotypes having significantly smaller CC areas. To investigate this, we defined an sSCA spectrum based on a descending Y:X karyotype ratio: 47,XYY, 46,XY, 48,XXYY, 47,XXY, 48,XXXY, 49,XXXXY, 46,XX, 47,XXX. We similarly explored the effects of both X and Y chromosome numbers within sex. Results of shape-based metrics were analyzed using permutation tests consisting of 5,000 iterations.

Results

Several subregional areas, local curvature, and BLDs differed between groups.Moderate associations were found between area and curvature in relation to the spectrum and X and Y chromosome counts. BLD was strongly associated with X chromosome count in both male and female groups.

Conclusions

Our results suggest that X- and Y-linked genes have differential effects on CC morphometry. To our knowledge, this is the first study to compare CC morphometry across these extremely rare groups.