Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

De novo sphingolipid synthesis is essential for viability, but not for transport of glycosylphosphatidylinositol-anchored proteins, in African trypanosomes

De novo sphingolipid synthesis is required for the exit of glycosylphosphatidylinositol (GPI)-anchored membrane proteins from the endoplasmic reticulum in yeast. Using a pharmacological approach, we test the generality of this phenomenon by analyzing the transport of GPI-anchored cargo in widely divergent eukaryotic systems represented by African trypanosomes and HeLa cells. Myriocin, which blocks the first step of sphingolipid synthesis (serine + palmitate --> 3-ketodihydrosphingosine), inhibited the growth of cultured bloodstream parasites, and growth was rescued with exogenous 3-ketodihydrosphingosine. Myriocin also blocked metabolic incorporation of [3H]serine into base-resistant sphingolipids. Biochemical analyses indicate that the radiolabeled lipids are not sphingomyelin or inositol phosphorylceramide, suggesting that bloodstream trypanosomes synthesize novel sphingolipids. Inhibition of de novo sphingolipid synthesis with myriocin had no adverse effect on either general secretory trafficking or GPI-dependent trafficking in trypanosomes, and similar results were obtained with HeLa cells. A mild effect on endocytosis was seen for bloodstream trypanosomes after prolonged incubation with myriocin. These results indicate that de novo synthesis of sphingolipids is not a general requirement for secretory trafficking in eukaryotic cells. However, in contrast to the closely related kinetoplastid Leishmania major, de novo sphingolipid synthesis is essential for the viability of bloodstream-stage African trypanosomes.     

Developmentally regulated sphingolipid synthesis in African trypanosomes

Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream-stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labelling confirmed stage-specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased more than threefold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six-transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian sphingomyelin synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites.     

Genetic dissection of Al tolerance QTLs in the maize genome by high density SNP scan

Background

Aluminum (Al) toxicity is an important limitation to food security in tropical and subtropical regions. High Al saturation on acid soils limits root development, reducing water and nutrient uptake. In addition to naturally occurring acid soils, agricultural practices may decrease soil pH, leading to yield losses due to Al toxicity. Elucidating the genetic and molecular mechanisms underlying maize Al tolerance is expected to accelerate the development of Al-tolerant cultivars.

Results

Five genomic regions were significantly associated with Al tolerance, using 54,455 SNP markers in a recombinant inbred line population derived from Cateto Al237. Candidate genes co-localized with Al tolerance QTLs were further investigated. Near-isogenic lines (NILs) developed for ZmMATE2 were as Al-sensitive as the recurrent line, indicating that this candidate gene was not responsible for the Al tolerance QTL on chromosome 5, qALT5. However, ZmNrat1, a maize homolog to OsNrat1, which encodes an Al³⁺ specific transporter previously implicated in rice Al tolerance, was mapped at ~40 Mbp from qALT5. We demonstrate for the first time that ZmNrat1 is preferentially expressed in maize root tips and is up-regulated by Al, similarly to OsNrat1 in rice, suggesting a role of this gene in maize Al tolerance. The strongest-effect QTL was mapped on chromosome 6 (qALT6), within a 0.5 Mbp region where three copies of the Al tolerance gene, ZmMATE1, were found in tandem configuration. qALT6 was shown to increase Al tolerance in maize; the qALT6-NILs carrying three copies of ZmMATE1 exhibited a two-fold increase in Al tolerance, and higher expression of ZmMATE1 compared to the Al sensitive recurrent parent. Interestingly, a new source of Al tolerance via ZmMATE1 was identified in a Brazilian elite line that showed high expression of ZmMATE1 but carries a single copy of ZmMATE1.

Conclusions

High ZmMATE1 expression, controlled either by three copies of the target gene or by an unknown molecular mechanism, is responsible for Al tolerance mediated by qALT6. As Al tolerant alleles at qALT6 are rare in maize, marker-assisted introgression of this QTL is an important strategy to improve maize adaptation to acid soils worldwide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-153) contains supplementary material, which is available to authorized users.     

Association of genetic variants rs641153 (CFB), rs2230199 (C3), and rs1410996 (CFH) with age-related macular degeneration in a Brazilian population

This study aimed to investigate the association among genetic variants of the complement pathway CFB R32Q (rs641153), C3 R102G (rs2230199), and CFH (rs1410996) with age-related macular degeneration (AMD) in a sample of the Brazilian population. In a case-control study, 484 AMD patients were classified according to the clinical age-related maculopathy grading system (CARMS) and compared to 479 unrelated controls. The genetic variants rs1410996 of complement H (CFH), rs641153 of complement factor B (CFB), and rs2230199 of complement 3 (C3) were evaluated through polymerase chain reaction (PCR) and direct sequencing. The associations between single nucleotide polymorphisms (SNPs) and AMD, adjusted by age, were assessed by using logistic regression models. A statistically significant association was observed between AMD risk and rs2230199 variant with an OR of 2.01 (P  = 0.0002) for CG individuals compared to CC individuals. Regarding the comparison of advanced AMD versus the control group, the OR was 2.12 (P = 0.0036) for GG versus AA genotypes for rs1410996 variant. Similarly, the OR for rs2230199 polymorphism was 2.3034 (P  = 5.47^e-05) when comparing CG individuals to CC carriers. In contrast, the rs641153 variant showed a significant protective effect against advanced AMD for GA versus GG genotype (OR = 0.4406; P  = 0.0019). When comparing wet AMD versus controls, a significant association was detected for rs1410996 variant (OR = 2.16; P  = 0.0039) comparing carriers of the homozygous GG versus AA genotype, as well as in the comparisons of GG (OR = 3.0713; P  = 0.0046) and CG genotypes (OR = 2.2249; P  = 0.0002) versus CC genotype for rs2230199 variant, respectively. The rs641153 variant granted a significant protective effect against wet AMD for GA versus GG genotypes (OR = 0.4601; P  = 0.0044). Our study confirmed the risk association between rs2230199 and rs1410996 variants and AMD, and the protective role against AMD for rs641153 variant.     

Role of AP-1 in Developmentally Regulated Lysosomal Trafficking in Trypanosoma brucei

African trypanosomes are the causative agents of human trypanosomiasis (sleeping sickness). The pathogenic stage of the parasite has unique adaptations to life in the bloodstream of the mammalian host, including upregulation of endocytic and lysosomal activities. We investigated stage-specific requirements for cytoplasmic adaptor/clathrin machinery in post-Golgi apparatus biosynthetic sorting to the lysosome using RNA interference silencing of the Tbμ1 subunit of adaptor complex 1 (AP-1), in conjunction with immunolocalization, kinetic analyses of reporter transport, and quantitative endocytosis assays. Tbμ1 silencing was lethal in both stages, indicating a critical function(s) for the AP-1 machinery. Transport of soluble and membrane-bound secretory cargoes was Tbμ1 independent in both stages. In procyclic parasites, trafficking of the lysosomal membrane protein, p67, was disrupted, leading to cell surface mislocalization. The lysosomal protease trypanopain was also secreted, suggesting a transmembrane-sorting receptor for this soluble hydrolase. In bloodstream trypanosomes, both p67 and trypanopain trafficking were unaffected by Tbμ1 silencing, suggesting that AP-1 is not necessary for biosynthetic lysosomal trafficking. Endocytosis in bloodstream cells was also unaffected, indicating that AP-1 does not function at the flagellar pocket. These results indicate that post-Golgi apparatus sorting to the lysosome is critically dependent on the AP-1/clathrin machinery in procyclic trypanosomes but that this machinery is not necessary in bloodstream parasites. We propose a simple model for stage-specific default secretory trafficking in trypanosomes that is consistent with the behavior of other soluble and glycosylphosphatidylinositol-anchored cargos and which is influenced by upregulation of endocytosis in bloodstream parasites as an adaptation to life in the mammalian bloodstream.African trypanosomes (Trypanosoma brucei subspecies), the agents of African sleeping sickness, are alone among the kinetoplastid parasites (including Trypanosoma cruzi and Leishmania spp.) in having a pathogenic bloodstream stage that exists and replicates extracellularly in the mammalian host. This places unique constraints on the parasite in terms of dealing with host immune responses and on acquisition of essential nutrients. The parasite has evolved many strategies to deal with these constraints, the best known of which is the process of antigenic variation (9). Another is the lysosome, which impacts the host-pathogen balance in multiple ways. Trypanosomes have a single terminal lysosome that is the final repository of endocytic cargo acquired from the host serum for nutritional purposes (30), as well as for potentially lytic immune complexes removed from the cell surface (4, 8). Both endocytosis and lysosomal hydrolytic activities are differentially regulated through the trypanosome life cycle (11, 30), and there are stage-specific differences in the biosynthetic trafficking of essential lysosomal components (discussed below). The release of lysosomal proteases is a factor in the signature event of human infection, penetration of the central nervous system (36). Finally, lysosomal physiology is critical to the activity of an innate human serum resistance trait, trypanolytic factor, which limits the host range of Trypanosoma species (38).Clearly, given its multiple roles in pathogenesis, biogenesis of the lysosome is critical to the success of trypanosomes as human parasites. As in all eukaryotes, lysosomal biogenesis is a balance between the proper sorting of newly synthesized membranes and proteins and recycling of established membranes and proteins internalized from the cell surface. In each case, protein sorting involves recognition of specific signals in cargo molecules by cellular machinery for inclusion in nascent transport vesicles destined for downstream delivery. Unique sets of cytoplasmic coat complexes at discrete intracellular locations serve the dual purpose of simultaneously mediating vesicle formation and selective cargo loading. The best characterized of these machineries is the clathrin/adaptin system for formation of coated vesicles at the Golgi apparatus and the plasma membrane (10, 41). Adaptor complexes (APs) are cytosolic heterotetramers that interact with specific signals in the cytoplasmic domains of membrane cargo proteins, such as dileucine motifs ([E/D]XXXL[L/I]) and tyrosine motifs (YXXØ, where Ø is a bulky hydrophobic residue). The prototypic AP complexes are AP-1 and AP-2, which function at the trans-Golgi network and plasma membrane, respectively. Both are composed of two large subunits (γ/β1 in AP-1; α/β2 in AP-2) and two smaller subunits (σ1/μ1 in AP-1; σ2/μ2 in AP-2). YXXØ motifs interact with μ adaptins, and dileucine motifs interact with combinations of adaptin subunits in both AP-1 and AP-2 (26, 40, 42). It is the large subunits, particularly β adaptin, that mediate clathrin recruitment (19, 44). Other APs, AP-3 and AP-4, with discrete subunit compositions, also exist. AP-3 functions in trafficking to lysosome-related organelles, such as melanosomes, and AP-4 may be involved in basolateral trafficking in polarized epithelial cells (10). The genome of the African trypanosome, T. brucei, encodes a complete complement of orthologous subunits for AP-1, AP-3, and AP-4 but has no genes for AP-2, the major adaptor complex mediating endocytosis in vertebrate cells (16). This is likely due to evolutionary loss, since the closely related T. cruzi has orthologues of all four APs.Two major lysosomal cargo proteins have been studied in T. brucei, the LAMP (lysosome-associated membrane protein)-like protein p67 and the cathepsin L orthologue trypanopain. p67 is a type I membrane protein with a large glycosylated lumenal domain and a short cytoplasmic domain (1, 27). In procyclic insect stage (PCF) trypanosomes, the cytoplasmic domain is both necessary and sufficient for lysosomal targeting of a heterologous reporter, and its deletion results in mistargeting of p67 to the cell surface (1). The cytoplasmic domain contains two canonical dileucine motifs, mutation of which also results in delivery to the cell surface (47). These findings strongly indicate the existence of cognate cytoplasmic machinery for lysosomal delivery of p67 in PCF trypanosomes. Strikingly, however, the cytoplasmic domain, and its motifs, are totally dispensable for lysosomal targeting in bloodstream stage (BSF) trypanosomes (1). Deletion of the cytoplasmic domain results in minor mislocalization to the cell surface, but p67 is still overwhelmingly delivered to the lysosome. Ongoing lysosomal targeting cannot easily be attributed to misfolding of the lumenal domain, as suggested by others (3), since the normal transport-associated patterns of p67 glycosylation and cleavage prevail in these deletion constructs.Less is known about targeting of soluble trypanopain. In mammalian cells, soluble hydrolases are targeted to the lysosome by the addition of mannose-6-phosphate (M6P) moieties in the Golgi apparatus, which serve as ligands for recognition and lysosomal targeting by downstream M6P receptors (28). Soluble hydrolases can also be sorted by receptors that recognize polypeptide motifs, such as sortilins in mammalian cells (12) and Vps10 in yeast (13, 32). These receptors have lumenal cargo recognition domains and cytoplasmic domains containing signals for late endosomal targeting and recycling. M6P-modified N-linked glycans are not found in trypanosomes, and genes encoding the necessary enzymatic activities are absent from the genome (16), ruling out this possibility for trypanopain sorting. However, the T. cruzi orthologue, cruzipain, has been shown to rely on peptide motifs in the N-terminal prodomain for targeting (24), raising the possibility of a sortilin/Vps10p-like sorting receptor. Although there are no obvious orthologues of these proteins in the T. brucei genome, overexpression of trypanopain in PCF trypanosomes leads to secretion, an observation that is consistent with saturation of a specific sorting receptor (S. S. Sutterwala and J. D. Bangs, unpublished observations).Having previously studied the innate signals involved in p67 targeting (1, 47), we now turned our attention to the cognate machinery for post-Golgi apparatus sorting. Specifically, we investigate the role of trypanosomal AP-1 in stage-specific biosynthetic trafficking to the lysosome using RNA interference (RNAi)-mediated silencing of the Tbμ1 (geneDB no. Tb927.7.3180 [www.genedb.org]) subunit as our primary strategy. Our results demonstrate that AP-1 and clathrin are critical for lysosomal targeting of p67 and trypanopain in PCF trypanosomes but that they are essentially dispensable in BSF parasites. These data, in conjunction with the behavior of p67-targeting mutants (1) and other trypanosomal secretory reporters, lead us to propose a simple model for stage-specific default trafficking in African trypanosomes. Although in some respects our results are similar to those of a recent publication using RNAi silencing of the Tbγ1 subunit of AP-1 (3), they differ in key aspects, leading us to significantly different conclusions.     

Cell cycle related proteins in hyperplasia of usual type in breast specimens of patients with and without breast cancer

Background  

Hyperplasia of usual type (HUT) is a common proliferative lesion associated with a slight elevated risk for subsequent development of breast cancer. Cell cycle-related proteins would be helpful to determine the putative role of these markers in the process of mammary carcinogenesis. The aim of this study was to analyze the expression of cell cycle related proteins in HUT of breast specimens of patients with and without breast cancer, and compare this expression with areas of invasive carcinomas.