Conserved TCP domain of Sas-4/CPAP is essential for pericentriolar material tethering during centrosome biogenesis

Pericentriolar material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4’s tethering role, we determined the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of β-sheets fold. This unique feature of the TCP domain suggests that it could provide an “extended surface-like” platform to tether the Sas-4–PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9–10th β-strands (β9–10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 β9–10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the β9–10 surface in mediating protein–protein interactions for efficient Sas-4–PCM scaffold centriole tethering. Hence, we provide possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication.Centrosomes consist of a pair of centrioles surrounded by a protein network of pericentriolar material (PCM), the main sites for microtubule nucleation and anchoring and thus responsible for PCM’s role as the principle microtubule-organizing centers (MTOCs) of cells (1–4). When PCM is not recruited, centrioles are unstable, and thus no functional centrosomes are generated (5, 6). Although initial proteomic studies suggested PCM to be an amorphous cloud composed of more than a 100 different proteins (7), recent superresolution microscopy of fly and human centrosomes have indicated key centrosomal proteins essential for centrosome biogenesis to be organized into distinct spatial compartments before appearing as a PCM cloud surrounding the centriole (6, 8–11).Thus, there could be a protein providing an interface for mediating PCM tethering to a centriole, a suitable candidate of which is the conserved centrosomal protein Sas-4 (CPAP in human), forming a layer closely associated with the centriole wall and yet shown to interact with various PCM components (6, 12). Functional studies in various model organisms suggest that Sas-4 proteins are required for both centriole formation and PCM assembly (6, 12); in the absence of Sas-4, nascent centrioles form but fail to mature into centrosomes (6). Overexpression of Sas-4 in flies produces PCM-like structures (13), whereas reduced amounts of Sas-4 in worms result in centrosomes having proportionally less PCM (12). Thus, although it is clear that Sas-4 is essential for centrosome biogenesis, the mechanisms by which Sas-4 contributes to PCM assembly remains elusive.During the course of these studies, we and others have reported that Sas-4/CPAP, a protein essential for centriole formation was found to interact with several centrosomal and PCM proteins including Cnn, Asl, D-PLP, γ-TuRC, SIL, Cep135, Cep120, and tubulin dimers (5, 6, 14–16). In Drosophila, the N-terminal domain of Sas-4 provides a scaffolding site for cytoplasmic protein complexes (hereafter referred to as Sas-4–PCM scaffold) and tethers the components of Sas-4–PCM scaffold to a centrosome matrix via its C terminus (6).Interestingly, the C-terminal region of Sas-4 proteins contains a conserved TCP10c domain (Pfam: PF07202) (hereafter referred to as TCP for brevity) (Fig. 1A and SI Appendix, Fig. S1). An E1235V missense mutation within this domain in CPAP has been identified in patients with primary microcephaly (MCPH), resulting in a reduced interaction with STIL (Ana2 in Drosophila), a centriole duplication factor also implicated in MCPH (16–18). Accordingly, recent structural studies on CPAP-STIL complex revealed that CPAP-STIL interaction is required during centriole assembly (19, 20). The C-terminal domain of CPAP has also been shown to mediate an interaction with another MCPH protein Cep135 (Bld-10 in Drosophila) and that interaction is required for centriole assembly. Bld-10 is a core centriolar protein and is required to stabilize structural integrity of centrioles (21–23). Taken together, we therefore speculate that the TCP domain could mediate protein–protein interactions and might serve as a tethering site for Sas-4–PCM scaffold–centriole interactions.Open in a separate windowFig. 1.Crystal structure of Drosophila Sas-4–TCP domain. (A) Domain architecture of Drosophila Sas-4 and its human ortholog CPAP. The fragment used for crystallization is indicated by a black underline. (B) Cartoon view of the overall structure of Sas-4–TCP. The invisible part of β16–20 in the crystal structure is shown as dotted lines. (C) Side view of Sas-4–TCP along the longitudinal axis from the N to C termini. Twisting of the TCP β-strands is diagramed below. F_L, surface left to β1; F_R, surface right to β1. (D and E) Cross-strand ladder residues on F_L (D) and F_R (E) are shown in spheres and classified into different types by color (purple, positively charged residues; red, negatively charged residues; orange, polar residues; green, hydrophobic residues).Although it appears that Sas-4 plays pivotal roles in centriole formation, assembling protein complexes in the cytoplasm, and tethering them to a developing centrosome, the mechanisms by which Sas-4 accomplishes its tethering role have remained unclear. In this study, we therefore investigated the structural basis of Sas-4 and show that via its conserved C-terminal TCP domain, it could provide an “extended surface-like” platform by which Sas-4 could mediate the Sas-4–PCM scaffold–centriole interaction during centrosome biogenesis.     

Ubiquitin-dependent degradation of multiple F-box proteins by an autocatalytic mechanism.

Ubiquitin-dependent degradation of regulatory proteins controls many cellular processes, including cell cycle progression, morphogenesis, and signal transduction. Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin ligases composed of a core complex including Skp1p, Cdc53p, one of multiple F-box proteins that are thought to provide substrate specificity to the complex, and the ubiquitin-conjugating enzyme, Cdc34p. It is not understood how SCF complexes are regulated and how physiological conditions alter their levels. Here we show that three F-box proteins, Grr1p, Cdc4p, and Met30p, are unstable components of the SCF, and are themselves degraded in a ubiquitin- and proteasome-dependent manner in vivo. Ubiquitination requires all the core components of the SCF and an intact F-box, suggesting that ubiquitination occurs within the SCF complex by an autocatalytic mechanism. Cdc4p and Grr1p are intrinsically unstable, and their steady-state levels did not fluctuate through the cell cycle. Taken together, our results suggest that ubiquitin-dependent degradation of F-box proteins allows rapid switching among multiple SCF complexes, thereby enabling cells to adapt quickly to changing physiological conditions and progression through different phases of the cell cycle.     

Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole

The acquisition of host-derived lipids is essential for the pathogenesis of the obligate intracellular bacteria Chlamydia trachomatis. Current models of chlamydial lipid acquisition center on the fusion of Golgi-derived exocytic vesicles and endosomal multivesicular bodies with the bacteria-containing parasitophorous vacuole ("inclusion"). In this study, we describe a mechanism of lipid acquisition and organelle subversion by C. trachomatis. We show by live cell fluorescence microscopy and electron microscopy that lipid droplets (LDs), neutral lipid storage organelles, are translocated from the host cytoplasm into the inclusion lumen. LDs dock at the surface of the inclusion, penetrate the inclusion membrane and intimately associate with reticulate Bodies, the replicative form of Chlamydia. The inclusion membrane protein IncA, but not other inclusion membrane proteins, cofractionated with LDs and accumulated in the inclusion lumen. Therefore, we postulate that the translocation of LDs may occur at IncA-enriched subdomains of the inclusion membrane. Finally, the chlamydial protein Lda3 may participate in the cooption of these organelles by linking cytoplasmic LDs to inclusion membranes and promoting the removal of the LD protective coat protein, adipocyte differentiation related protein (ADRP). The wholesale transport of LDs into the lumen of a parasitophorous vacuole represents a unique mechanism of organelle sequestration and subversion by a bacterial pathogen.     

The functional repressor parts of a tetrameric lac repressor-beta-galactosidase chimaera are organized as dimers.

The chimaeric protein repressor-galactosidase, in which fully active lac repressor is covalently linked to the active enzyme beta-galactosidase, was used as a system for probing the quaternary structure of lac repressor. Electron micrographs revealed repressor-galactosidase to be a tetrameric aggregate. When lac repressor, alone, was crosslinked with dimethyl suberimidate, dimers, trimers, tetramers, and oligomers of the protein subunit were produced, whereas crosslinking of the tetrameric repressor-galactosidase resulted in the production of only dimers of the chimaera. Treatment of lac repressor with iodine resulted in the formation of protein dimers; the same result was obtained with repressor-galactosidase. After limited proteolysis of lac repressor, no crosslinking was obtained after treatment with dimethyl suberimidate, whereas iodine still produced a covalent linkage. These results are interpreted as evidence that the lac repressor parts of the tetrameric repressor-galactosidase-chimaera are organized as dimers on the tetrameric-beta-galactosidase core. Because this chimaera has been previously shown to have normal repressor activity [B. Müller-Hill and J. Kania (1974) Nature, 249,561-563], we conclude that lac repressor still is biologically active as a dimeric aggregate.     

Fibrin structure in organized thrombotic material removed during pulmonary artery endarterectormy: the effect of vessel calibre

Pulmonary endarterectomy (PEA) is a curative therapeutic approach in patients with chronic thromboembolic pulmonary hypertension (CTEPH). The location-dependent structural differences of thrombotic material found in pulmonary arteries in CTEPH are poorly investigated. We present the case of a 47-year-old woman with antiphospholipid syndrome, diabetes mellitus and abnormal fibrin phenotype, who underwent PEA for CTEPH. Intravascular material removed bilaterally during PEA (from lobar, segmental and sub-segmental arteries) has been studied using light and scanning electron microscopy (SEM). Light microscopy showed tighter fibrous network in the portions of intraluminal thrombotic material facing the vessel wall, which contained collagen and fibrin fibers, and abundant cells. Cells, evaluated by immunostaining, were present in the whole removed material. Tissue factor expression was also observed with the highest values in the portions of intravascular material facing the vessel wall. In the main pulmonary arteries, SEM images revealed thick fibers of fibrous proteins loosly meshed and few erythrocytes and platelets between them (both dysmorphic “wedged” and fresh cells were present). In the fibrotic layers, containing mainly collagen and fibrin, removed from the lobar/segmental pulmonary arteries we found a stepwise increase in fiber density with decreasing vessel calibre, followed by denser fibrous networks composed of thinner fibers. Elastic fibers in the lobar and segmental arteries were aligned along the blood flow vector. These findings demonstrate differences in the structure of endarterectomized PEA material dependent on the vessel calibre and might contribute to understanding of CTEPH pathophysiology.     

Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released

Platelets, in addition to their function in hemostasis, play an important role in wound healing and tumor growth. Because platelets contain angiogenesis stimulators and inhibitors, the mechanisms by which platelets regulate angiogenesis remain unclear. As platelets adhere to activated endothelium, their action can enhance or inhibit local angiogenesis. We therefore suspected a higher organization of angiogenesis regulators in platelets. Using double immunofluorescence and immunoelectron microscopy, we show that pro- and antiangiogenic proteins are separated in distinct subpopulations of alpha-granules in platelets and megakaryocytes. Double immunofluorescence labeling of vascular endothelial growth factor (VEGF) (an angiogenesis stimulator) and endostatin (an angiogenesis inhibitor), or for thrombospondin-1 and basic fibroblast growth factor, confirms the segregation of stimulators and inhibitors into separate and distinct alpha-granules. These observations motivated the hypothesis that distinct populations of alpha-granules could undergo selective release. The treatment of human platelets with a selective PAR4 agonist (AYPGKF-NH(2)) resulted in release of endostatin-containing granules, but not VEGF-containing granules, whereas the selective PAR1 agonist (TFLLR-NH(2)) liberated VEGF, but not endostatin-containing granules. In conclusion, the separate packaging of angiogenesis regulators into pharmacologically and morphologically distinct populations of alpha-granules in megakaryocytes and platelets may provide a mechanism by which platelets can locally stimulate or inhibit angiogenesis.     

Elongating nerve fibers are guided by a pathway of material released from embryonic nonneuronal cells.

Dissociated parasympathetic neurons rapidly initiate neurite outgrowth when exposed to culture medium previously conditioned by the growth of embryonic heart cells. The inducer of neurite outgrowth in the conditioned medium is a substratum-conditioning factor; that is, it does not act in a soluble form, but acts only when bound to the nerve cell culture substratum. When a sharp border is created between a region of the substratum coated with this factor and a region coated with unconditioned medium, neurites fail to cross this border; rather, they change their direction of outgrowth so as to remain on the conditioned substratum. Thus, long after the initiation of outgrowthhas been induced, elongating neurites continue to respond to the substratum-conditioning factor in a manner that allows their outgrowth to be channeled along a pathway of this neurotropic substratum-associated material.     

The ovine sexually dimorphic nucleus of the medial preoptic area is organized prenatally by testosterone

A sexually dimorphic nucleus that can be identified in adult sheep by its characteristic pattern of cytochrome P450 aromatase mRNA exists in the preoptic/anterior hypothalamic area and is called the ovine sexually dimorphic nucleus (oSDN). In other species, male-typical sexually dimorphic preoptic nuclei develop under the influence of gonadal testosterone exposure. Thus, we hypothesized that the oSDN develops before birth in the sheep and is organized by exposure to testosterone. To test this, we determined whether an identifiable oSDN is present in the fetal lamb brain at d 130-140 gestation (term approximately 150 d). In situ hybridization for aromatase mRNA revealed a cell group in the caudal preoptic area that corresponded to the oSDN in adults. Quantitative analysis showed that the mean volume of the oSDN in late-gestation fetuses was significantly larger in male than in female lamb fetuses. We next treated a group of pregnant ewes with testosterone propionate (TP) from d 30-90 gestation and measured oSDN volumes in TP-exposed and control fetuses on d 135 gestation. The mean volume of the oSDN in female fetuses exposed to TP was significantly larger than in control females and also larger than in control and TP-exposed males. Taken together, these data demonstrate that testosterone acts during a prenatal critical period to organize the development of aromatase-expressing neurons into the male-typical oSDN in sheep.     

Chiral symmetry breaking by spatial confinement in tactoidal droplets of lyotropic chromonic liquid crystals

In many colloidal systems, an orientationally ordered nematic (N) phase emerges from the isotropic (I) melt in the form of spindle-like birefringent tactoids. In cases studied so far, the tactoids always reveal a mirror-symmetric nonchiral structure, sometimes even when the building units are chiral. We report on chiral symmetry breaking in the nematic tactoids formed in molecularly nonchiral polymer-crowded aqueous solutions of low-molecular weight disodium cromoglycate. The parity is broken by twisted packing of self-assembled molecular aggregates within the tactoids as manifested by the observed optical activity. Fluorescent confocal microscopy reveals that the chiral N tactoids are located at the boundaries of cells. We explain the chirality induction as a replacement of energetically costly splay packing of the aggregates within the curved bipolar tactoidal shape with twisted packing. The effect represents a simple pathway of macroscopic chirality induction in an organic system with no molecular chirality, as the only requirements are orientational order and curved shape of confinement.     

Purified preparations of human luteinizing hormone are contaminated with small amounts of a chorionic gonadotropin-like material

We have identified a CG-like protein contaminating a purified human LH preparation of immunochemical grade. This CG-like material is estimated to comprise 0.17%, by weight, of the LH and reacts in specific, sequential-type, two-monoclonal antibody, immunoradiometric assays for CG as well as in the carboxyl-tail CG RIA. The CG-like material is not separable from LH by size exclusion or ion exchange chromatography. The LH can be freed of this small contamination of CG-like material by immunopurification employing specific monoclonal antibodies. Sephadex G-100 chromatography shows this material to have a mol wt of 40.0K. Western blot analysis of the LH run under nonreducing conditions, using an anti-CG carboxyl-tail primary antibody, reveals two bands of this CG-like material, one at 60.8K and one at 50.7K. When electrophoresed under reducing conditions, the material reacts with the anti-CG carboxyl-tail antibody at several mol wt, ranging from 10.5-64K.     

Dried cytospin preparations of synovial fluid are a stable material for long-time storage and delayed crystal analysis

The aim of this study was to evaluate dried SF cytospin preparations as a suitable medium for long-time storage and delayed crystal analysis. For this purpose, we analyzed ten MSU-positive, ten CPPD-positive and 20 crystal-negative SF at baseline (wet preparation), after 24 h, 1 week, 4 weeks, 6 months and 12 months for the occurrence of crystals. After cytocentrifugation for 10 min at 700 rpm in a Shandon Cytospin 4 cytocentrifuge (Thermo Fisher Scientific, Waltham, USA), the sediments were dried on the slides and examined in blinded fashion at any time point by an experienced analyst using polarized microscopy. The crystal content of the initially MSU- and CPPD-positive samples was positively confirmed at any time point of the study, whereas the controls remained crystal-negative during the whole study period. Thus, compared to the examined wet preparations at baseline, there were no false positive or false negative results observed. In conclusion, dried cytospin preparations were confirmed as a suitable material for long-time storage and delayed crystal identification.     

Induction of lipid droplets in fibroblasts of the hamster lung by a diet high in vitamin A

To investigate the effect of vitamin A on the occurrence of lipid droplets in alveolar fibroblasts, light and electron microscopic studies were carried out on the lungs of Syrian golden hamsters fed on diets with a low, normal, or high vitamin A content for a period of 17 weeks. The feeding of the high vitamin A diet resulted in a considerable increase in size and number of lipid droplets in alveolar fibroblasts. These droplets also occurred in fibroblasts of the visceral pleura, and in fibroblasts of connective tissue around bronchioli and larger blood vessels. Most of the lipid-laden fibroblasts could be recognized as myofibroblasts. The significance of the droplets for the deposition of interstitial materials or for the synthesis of prostaglandins is discussed.     

Immunosuppression by surface-active material: lack of species specificity

Surface-active material isolated from the lungs of both dogs and rats was tested for its ability to suppress the in vitro proliferative responses of dog, mouse, or human lymphocytes to a variety of immunologic stimuli. Both dog and rat surface-active material exerted a dose-dependent suppressive effect on the proliferative responses of each species of lymphocyte, regardless of the nature of the immune stimulus (mitogen, antigen, or alloantigen). The data indicated that surface-active material acts by directly inhibiting the responding lymphocyte and not by activating suppessor cells. The immunosuppression could not be attributed to lymphocyte cytotoxicity. Although the mechanism of this immunosuppressive action of surface-active material remains undefined, the present data clearly indicate that such activity is not species specific.