Coiled-coil Protein Research Articles

Involvement of the centrosomal protein 55 (cep55) gene in zebrafish head formation.

Mammalian CEP55 (centrosomal protein 55kDa) is a coiled-coil protein localized to the centrosome in interphase cells and is required for cytokinesis. A homozygous non-sense mutation in human CEP55 has been recently identified in perinatal lethal MARCH (multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia and hydranencephaly) syndrome. We have isolated zebrafish cep55 mutants defective in head morphology. The zebrafish cep55 gene was expressed in the head including the retina and the pectoral fin at 1day post-fertilization (dpf), and extensive cell death was widely observed in the head and tail of the cep55 mutant. In the cep55 mutant, the anterior-posterior distance of the ventral pharyngeal arches was short, and retinal lamination was disorganized. Neural cells, such as islet1-positive cells and pax2-positive cells, and fli1b-positive vascular cells were reduced in the head of the cep55 mutant. Thus, we propose that the zebrafish cep55 mutant is a model organism for human MARCH syndrome.

The Golgin Protein Giantin Regulates Interconnections Between Golgi Stacks.

Golgins are a family of Golgi-localized long coiled-coil proteins. The major golgin function is thought to be the tethering of vesicles, membranes, and cytoskeletal elements to the Golgi. We previously showed that knockdown of one of the longest golgins, Giantin, altered the glycosylation patterns of cell surfaces and the kinetics of cargo transport, suggesting that Giantin maintains correct glycosylation through slowing down transport within the Golgi. Giantin knockdown also altered the sizes and numbers of mini Golgi stacks generated by microtubule de-polymerization, suggesting that it maintains the independence of individual Golgi stacks. Therefore, it is presumed that Golgi stacks lose their independence following Giantin knockdown, allowing easier and possibly increased transport among stacks and abnormal glycosylation. To gain structural insights into the independence of Golgi stacks, we herein performed electron tomography and 3D modeling of Golgi stacks in Giantin knockdown cells. Compared with control cells, Giantin-knockdown cells had fewer and smaller fenestrae within each cisterna. This was supported by data showing that the diffusion rate of Golgi membrane proteins is faster in Giantin-knockdown Golgi, indicating that Giantin knockdown structurally and functionally increases connectivity among Golgi cisternae and stacks. This increased connectivity suggests that contrary to the cis-golgin tether model, Giantin instead inhibits the tether and fusion of nearby Golgi cisternae and stacks, resulting in transport difficulties between stacks that may enable the correct glycosylation of proteins and lipids passing through the Golgi.

Proteomics and bioinformatics approaches for the identification of plasma biomarkers to detect Parkinson's disease.

The aim of the present study was to screen for biomarkers of Parkinson's disease (PD) using proteomics and bioinformatics approaches. PD patients were divided into three groups: Those without surgery (PD1 group); those who had undergone deep brain stimulation (DBS) surgery without electrode stimulation (PD2 group); and those who had undergone DBS surgery with 1 month of electrode stimulation (PD3 group). The non-Parkinson control group (CK group) was also involved. Quantitative proteomic analysis of human sera was performed through the use of tandem mass tag markers and liquid chromatography-mass spectrometry (LC-MS)-based techniques. For the proteins with quantitative information, a systematic bioinformatics analysis was then performed, including protein annotation, functional classification, functional enrichment and cluster analysis based on functional enrichment. Of the 739 proteins identified, quantitative information was available for 644. With regard to differential expression, 18 upregulated and 21 downregulated proteins were screened in the PD1/CK comparison group; 12 upregulated and 12 downregulated proteins in the PD2/PD1 comparison group; and 16 upregulated and 19 downregulated proteins in the PD3/PD2 comparison group. Coiled-coil domain-containing protein 154 (CCDC154) and tripartite motif-containing protein 3 (TRIM3) were key proteins involved in the molecular mechanisms of PD, participating in intracellular vesicle, ubiquitin protein ligase and transition metal ion-binding activities. After DBS surgery, desert hedgehog protein (DHH) was downregulated, whereas neuropilin-2 (NRP2) was upregulated; these participated in the ensheathment of neurons and the semaphorin receptor complex, respectively. The expression level of chloride intracellular channel protein 1 (CLIC1) was increased after 1 month of electrode stimulation following DBS. By combining proteomic approaches and LC-MS methods, significant proteins including CCDC154, TRIM3, DHH, NRP2 and CLIC1 were detected with high specificity and sensitivity. These may be used as novel biomarkers for early diagnosis of PD and the future development of treatments.

Abstract 363: Systems Network Genomic Analysis Reveals the Role Of MURC/Cavin-4 in Cardiac Ischemia/reperfusion Injury

Background: Ischemia/reperfusion (I/R) injury is a critical issue in the development of treatment strategies for ischemic heart disease. Muscle-restricted coiled-coil protein (MURC)/Cavin-4, which is a component of caveolae, is involved in the pathophysiology of dilated cardiomyopathy and cardiac hypertrophy. However, the role of MURC in cardiac I/R injury remains unknown. Objective: To elucidate the role of MURC in cardiac I/R injury. Methods and Results: The systems network genomic analysis based on PC-corr network inference on microarray data between wild-type and MURC knockout (KO) mouse hearts described a network of discriminating genes associated with reactive oxygen species (ROS). To demonstrate the prediction, we investigated the effect of MURC deletion in cardiac I/R injury. MURC deletion in IR-injured mouse hearts decreased infarct size and preserved heart contraction with the inhibition of ROS production and ROS-related gene expressions such as EGR1 and DDIT4 as well as EGR1 protein level. PC-corr network inference integrated with a protein-protein interaction network prediction showed that MURC is also involved in the apoptotic pathway. We confirmed the upregulated activity of STAT3, which is a transcription factor of anti-apoptotic signaling, with the increase of BCL2 mRNA expression and protein level and the decrease of cleaved Caspase 3 protein level in MURC KO compared with WT mouse hearts after I/R. TUNEL assay showed that MURC modulates the apoptosis in cardiomyocytes exposed to hydrogen peroxide. STAT3 inhibitor cancelled the cardioprotective effect of MURC deletion in I/R-injured heart and the anti-apoptotic effect of MURC knockdown in cardiomyocytes. Conclusions: Our findings suggest that MURC plays a pivotal role in the regulation of ROS-induced cell death and STAT3-meditated anti-apoptotic signaling in cardiac I/R injury. MURC may be a therapeutic target for cardiac I/R injury.

Functional loss of Ccdc151 leads to hydrocephalus in a mouse model of primary ciliary dyskinesia.

ABSTRACTPrimary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder affecting normal structure and function of motile cilia, phenotypically manifested as chronic respiratory infections, laterality defects and infertility. Autosomal recessive mutations in genes encoding for different components of the ciliary axoneme have been associated with PCD in humans and in model organisms. The CCDC151 gene encodes for a coiled-coil axonemal protein that ensures correct attachment of outer dynein arm (ODA) complexes to microtubules. A correct arrangement of dynein arm complexes is required to provide the proper mechanical force necessary for cilia beat. Loss-of-function mutations in CCDC151 in humans leads to PCD disease with respiratory distress and defective left-right body asymmetry. In mice with the Ccdc151Snbl loss-of-function mutation (Snowball mutant), left-right body asymmetry with heart defects have been observed. Here, we demonstrate that loss of Ccdc151 gene function via targeted gene deletion in mice leads to perinatal lethality and congenital hydrocephalus. Microcomputed tomography (microCT) X-ray imaging of Ccdc151–β-galactosidase reporter expression in whole-mount brain and histological analysis show that Ccdc151 is expressed in ependymal cells lining the ventricular brain system, further confirming the role of Ccdc151 dysfunction in hydrocephalus development. Analyzing the features of hydrocephalus in the Ccdc151-knockout animals by microCT volumetric imaging, we observe continuity of the aqueduct of Sylvius, indicating the communicating nature of hydrocephalus in the Ccdc151-knockout animals. Congenital defects in left-right asymmetry and male infertility have been also observed in Ccdc151-null animals. Ccdc151 gene deletion in adult animals results in abnormal sperm counts and defective sperm motility.This article has an associated First Person interview with the joint first authors of the paper.

Stick-slip kinetics in a bistable bar immersed in a heat bath

Structural transitions in some rod-like biological macromolecules under tension are known to proceed by the propagation through the length of the molecule of an interface separating two phases. A continuum mechanical description of the motion of this interface, or phase boundary, takes the form of a kinetic law which relates the thermodynamic driving force across it with its velocity in the reference configuration. For biological macromolecules immersed in a heat bath, thermally activated kinetics described by the Arrhenius law is often a good choice. Here we show that ‘stick-slip’ kinetics, characteristic of friction, can also arise in an overdamped bistable bar immersed in a heat bath. To mimic a rod-like biomolecule we model the bar as a chain of masses and bistable springs moving in a viscous fluid. We conduct Langevin dynamics calculations on the chain and extract a temperature dependent kinetic relation by observing that the dissipation at a phase boundary can be estimated by performing an energy balance. Using this kinetic relation we solve boundary value problems for a bistable bar immersed in a constant temperature bath and show that the resultant force-extension relation matches very well with the Langevin dynamics results. We estimate the force fluctuations at the pulled end of the bar due to thermal kicks from the bath by using a partition function. We also show rate dependence of hysteresis in cyclic loading of the bar arising from the stick-slip kinetics. Our kinetic relation could be applied to rod-like biomolecules, such as, DNA and coiled-coil proteins which exhibit structural transitions that depend on both temperature and loading rate.

Thermoresponsive Protein-Engineered Coiled-Coil Hydrogel for Sustained Small Molecule Release.

Thermoresponsive hydrogels are used for an array of biomedical applications. Lower critical solution temperature-type hydrogels have been observed in nature and extensively studied in comparison to upper critical solution temperature (UCST)-type hydrogels. Of the limited protein-based UCST-type hydrogels reported, none have been composed of a single coiled-coil domain. Here, we describe a biosynthesized homopentameric coiled-coil protein capable of demonstrating a UCST. Microscopy and structural analysis reveal that the hydrogel is stabilized by molecular entanglement of protein nanofibers, creating a porous matrix capable of binding the small hydrophobic molecule, curcumin. Curcumin binding increases the α-helical structure, fiber entanglement, mechanical integrity, and thermostability, resulting in sustained drug release at physiological temperature. This work provides the first example of a thermoresponsive hydrogel comprised of a single coiled-coil protein domain that can be used as a vehicle for sustained release and, by demonstrating UCST-type behavior, shows promise in forging a relationship between coiled-coil protein-phase behavior and that of synthetic polymer systems.

GemC1 is a critical switch for neural stem cell generation in the postnatal brain.

The subventricular zone (SVZ) is one of two main niches where neurogenesis persists during adulthood, as it retains neural stem cells (NSCs) with self-renewal capacity and multi-lineage potency. Another critical cellular component of the niche is the population of postmitotic multiciliated ependymal cells. Both cell types are derived from radial glial cells that become specified to each lineage during embryogenesis. We show here that GemC1, encoding Geminin coiled-coil domain-containing protein 1, is associated with congenital hydrocephalus in humans and mice. Our results show that GemC1 deficiency drives cells toward a NSC phenotype, at the expense of multiciliated ependymal cell generation. The increased number of NSCs is accompanied by increased levels of proliferation and neurogenesis in the postnatal SVZ. Finally, GemC1-knockout cells display altered chromatin organization at multiple loci, further supporting a NSC identity. Together, these findings suggest that GemC1 regulates the balance between NSC generation and ependymal cell differentiation, with implications for the pathogenesis of human congenital hydrocephalus.

A role of GCC88 in the retrograde transport of CI-M6PR and the maintenance of lysosomal activity.

GCC88 is a golgin coiled-coil protein at the trans-Golgi (TGN) that functions as a tethering factor for the endosome-derived retrograde transport vesicles. Here, we demonstrate that GCC88 is required for the endosome-to-TGN retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-M6PR). The knockout of GCC88 perturbs the retrieval of CI-M6PR and decreases its cellular level at the steady state, which causes the improper processing of newly synthesized cathepsin-D, a lysosomal hydrolase dependent on CI-M6PR for its delivery to lysosomes. At the whole cell level, the knockout of GCC88 reduces the lysosomal proteolytic capacity but does not impair of the efficiency of autophagy within these cells.

CHCHD4 regulates tumour proliferation and EMT-related phenotypes, through respiratory chain-mediated metabolism

BackgroundMitochondrial oxidative phosphorylation (OXPHOS) via the respiratory chain is required for the maintenance of tumour cell proliferation and regulation of epithelial to mesenchymal transition (EMT)-related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study, we interrogate the importance of CHCHD4-regulated mitochondrial metabolism for tumour cell proliferation and EMT-related phenotypes, and elucidate key pathways involved.ResultsUsing in silico analyses of 967 tumour cell lines, and tumours from different cancer patient cohorts, we show that CHCHD4 expression positively correlates with OXPHOS and proliferative pathways including the mTORC1 signalling pathway. We show that CHCHD4 expression significantly correlates with the doubling time of a range of tumour cell lines, and that CHCHD4-mediated tumour cell growth and mTORC1 signalling is coupled to respiratory chain complex I (CI) activity. Using global metabolomics analysis, we show that CHCHD4 regulates amino acid metabolism, and that CHCHD4-mediated tumour cell growth is dependent on glutamine. We show that CHCHD4-mediated tumour cell growth is linked to CI-regulated mTORC1 signalling and amino acid metabolism. Finally, we show that CHCHD4 expression in tumours is inversely correlated with EMT-related gene expression, and that increased CHCHD4 expression in tumour cells modulates EMT-related phenotypes.ConclusionsCHCHD4 drives tumour cell growth and activates mTORC1 signalling through its control of respiratory chain mediated metabolism and complex I biology, and also regulates EMT-related phenotypes of tumour cells.

Artificial coiled coil biomineralisation protein for the synthesis of magnetic nanoparticles

Green synthesis of precise inorganic nanomaterials is a major challenge. Magnetotactic bacteria biomineralise magnetite nanoparticles (MNPs) within membrane vesicles (magnetosomes), which are embedded with dedicated proteins that control nanocrystal formation. Some such proteins are used in vitro to control MNP formation in green synthesis; however, these membrane proteins self-aggregate, making their production and use in vitro challenging and difficult to scale. Here, we provide an alternative solution by displaying active loops from biomineralisation proteins Mms13 and MmsF on stem-loop coiled-coil scaffold proteins (Mms13cc/MmsFcc). These artificial biomineralisation proteins form soluble, stable alpha-helical hairpin monomers, and MmsFcc successfully controls the formation of MNP when added to magnetite synthesis, regulating synthesis comparably to native MmsF. This study demonstrates how displaying active loops from membrane proteins on coiled-coil scaffolds removes membrane protein solubility issues, while retains activity, enabling a generic approach to readily-expressible, versatile, artificial membrane proteins for more accessible study and exploitation.

Overexpression of trans-Golgi network t-SNAREs rescues vacuolar trafficking and TGN morphology defects in a putative tethering factor mutant.

The trans-Golgi network (TGN) is a major site for sorting of cargo to either the vacuole or apoplast. The TGN-localized coiled-coil protein TNO1 is a putative tethering factor that interacts with the TGN t-SNARE SYP41 and is required for correct localization of the SYP61 t-SNARE. An Arabidopsis thaliana tno1 mutant is hypersensitive to salt stress and partially mislocalizes vacuolar proteins to the apoplast, indicating a role in vacuolar trafficking. Here, we show that overexpression of SYP41 or SYP61 significantly increases SYP41-SYP61 complex formation in a tno1 mutant, and rescues the salt sensitivity and defective vacuolar trafficking of the tno1 mutant. The TGN is disrupted and vesicle budding from Golgi cisternae is reduced in the tno1 mutant, and these defects are also rescued by overexpression of SYP41 or SYP61. Our results suggest that the trafficking and Golgi morphology defects caused by loss of TNO1 can be rescued by increasing SYP41-SYP61 t-SNARE complex formation, implicating TNO1 as a tethering factor mediating efficient vesicle fusion at the TGN.

106-OR: Geminin Expression in Human Fetal Pancreas

Geminin (Gmnn) is a coiled-coil nuclear protein originally identified as a DNA replication inhibitor. In addition, nuclear Gmnn regulates gene expression by interacting (through a domain distinct from the Cdt1-interacting domain) with Brg1, the catalytic subunit of the SWI/SNF chromatin remodeling complex. In this context, Gmnn regulates expression of key transcription factors involved in differentiation. The dual role of Gmnn in the cell cycle and differentiation has been viewed as a critical mechanism for linking replication and differentiation. We wanted to test its role in human pancreas development. We found a robust Gmnn expression in the human fetal pancreas but its expression slightly decreased with age. Remarkably the fraction of cells expressing both Gmnn and Ki67 declined much faster with age. We isolated 13WD human fetal pancreas and studied the proportion of replicating Sox9+cells. We compared control human fetal explants with fetal explants transduced with a shRNA lentivirus directed against Gmnn (Gmnnsh) and discovered that Gmnnshexplants display a decrease in proliferation compared to controls after 2 days in culture. RNA sequencing comparing Gmnnshexplants to controls revealed a marked decrease in transcripts associated with endocrine differentiation. To evaluate the effects on endocrine differentiation, human fetal pancreases were isolated at 13WD and treated for 5 days with lentiviruses or casein kinase inhibitors. We detected a significant reduction in the number of insulin+cells after treatment with the casein kinase inhibitor TBB and a significant reduction in the number of glucagon+cells after treatment with TBB or after siCsnk2a1. Overexpression of Gmnn significantly increased the proportion of Neurog3+cells compared to the control explants. Finally, we found that inhibition of Gmnn or casein kinase 2 was capable of significantly reducing expression of Nkx6.1 compared to controls. We conclude that Gmnn may play a critical role pancreas cell replication and endocrine specification in human development. Disclosure L. Baeyens: None. D.W. Scheel: None. K. Yang: None. M. German: Advisory Panel; Self; Encellin. Stock/Shareholder; Self; Encellin, Viacyte, Inc. Funding JDRF

Conserved juxtamembrane domains in the yeast golgin Coy1 drive assembly of a megadalton-sized complex and mediate binding to tethering and SNARE proteins

The architecture and organization of the Golgi complex depend on a family of coiled-coil proteins called golgins. Golgins are thought to form extended homodimers that are C-terminally anchored to Golgi membranes, whereas their N termini extend into the cytoplasm to initiate vesicle capture. Previously, we reported that the Saccharomyces cerevisiae golgin Coy1 contributes to intra-Golgi retrograde transport and binds to the conserved oligomeric Golgi (COG) complex and multiple retrograde Golgi Q-SNAREs (where SNARE is soluble NSF-attachment protein receptor). Here, using various engineered yeast strains, membrane protein extraction and fractionation methods, and in vitro binding assays, we mapped the Coy1 regions responsible for these activities. We also report that Coy1 assembles into a megadalton-size complex and that assembly of this complex depends on the most C-terminal coiled-coil and a conserved region between this coiled-coil and the transmembrane domain of Coy1. We found that this conserved region is necessary and sufficient for binding the SNARE protein Sed5 and the COG complex. Mutagenesis of conserved arginine residues within the C-terminal coiled-coil disrupted oligomerization, binding, and function of Coy1. Our findings indicate that the stable incorporation of Coy1 into a higher-order oligomer is required for its interactions and role in maintaining Golgi homeostasis. We propose that Coy1 assembles into a docking platform that directs COG-bound vesicles toward cognate SNAREs on the Golgi membrane.

A molecular model for self-assembly of the synaptonemal complex protein SYCE3

The synaptonemal complex (SC) is a supramolecular protein assembly that mediates homologous chromosome synapsis during meiosis. This zipper-like structure assembles in a continuous manner between homologous chromosome axes, enforcing a 100-nm separation along their entire length and providing the necessary three-dimensional framework for cross-over formation. The mammalian SC comprises eight components—synaptonemal complex protein 1–3 (SYCP1–3), synaptonemal complex central element protein 1–3 (SYCE1–3), testis-expressed 12 (TEX12), and six6 opposite strand transcript 1 (SIX6OS1)—arranged in transverse and longitudinal structures. These largely α-helical, coiled-coil proteins undergo heterotypic interactions, coupled with recursive self-assembly of SYCP1, SYCE2–TEX12, and SYCP2–SYCP3, to achieve the vast supramolecular SC structure. Here, we report a novel self-assembly mechanism of the SC central element component SYCE3, identified through multi-angle light scattering and small-angle X-ray scattering (SAXS) experiments. These analyses revealed that SYCE3 adopts a dimeric four-helical bundle structure that acts as the building block for concentration-dependent self-assembly into a series of discrete higher-order oligomers. We observed that this is achieved through staggered lateral interactions between self-assembly surfaces of SYCE3 dimers and through end-on interactions that likely occur through intermolecular domain swapping between dimer folds. These mechanisms are combined to achieve potentially limitless SYCE3 assembly, particularly favoring formation of dodecamers of three laterally associated end-on tetramers. Our findings extend the family of self-assembling proteins within the SC and reveal additional means for structural stabilization of the SC central element.

Molecular dynamics simulation of tropomyosin bound to actins/myosin in the closed and open states.

Tropomyosin (Tpm) is a dimeric coiled-coil protein that binds to filamentous actin, and regulates actin-myosin interaction by moving between three positions corresponding to the blocked, closed, and open states. To elucidate how Tpm undergoes transitions between these functional states, we have built structural models and conducted extensive molecular dynamics simulations of the Tpm-actins/myosin complex in the closed and open states (total simulation time >1.4 μs). Based on the simulation trajectories, we have analyzed the dynamics and energetics of a truncated Tpm interacting with actins/myosin under the physiological conditions. Our simulations have shown distinct dynamics of four Tpm periods (P3-P6), featuring pronounced biased fluctuations of P4 and P5 toward the open position in the closed state, which is consistent with a conformational selection mechanism for Tpm-regulated myosin binding. Additionally, we have identified key residues of Tpm specifically binding to actins/myosin in the closed and open state. Some of them were validated as functionally important in comparison with past functional/clinical studies, and the rest will make promising targets for future mutational experiments.

Thermal unfolding of various human non-muscle isoforms of tropomyosin

Tropomyosin (Tpm) is an α-helical coiled-coil protein dimer, which forms a continuous head-to-tail polymer along the actin filament. In striated muscles, Tpm plays an important role in the Ca2+-dependent regulation of muscle contraction. However, little is known about functional and especially structural properties of the numerous non-muscle Tpm isoforms. In the present work, we have applied circular dichroism (CD) and differential scanning calorimetry (DSC) to investigate thermal unfolding and domain structure of various non-muscle human Tpm isoforms. These isoforms, the products of two different genes, TPM1 and TPM3, also significantly differ by alternatively spliced exons: N-terminal exons 1a2b or 1b, internal exons 6a or 6b, and C-terminal exons 9a, 9c or 9d. Our results clearly demonstrate that structural properties of various non-muscle Tpm isoforms can be quite different depending on the presence of different alternatively spliced exons in their genes. These data show for the first time a significant difference in the thermal unfolding between muscle and non-muscle Tpm isoforms and indicate that replacement of alternatively spliced exons alters the stability of certain domains in the Tpm molecule.

Arabidopsis FLL2 promotes liquid–liquid phase separation of polyadenylation complexes

An important component of cellular biochemistry is the concentration of proteins and nucleic acids in non-membranous compartments1,2. These biomolecular condensates are formed from processes that include liquid-liquid phase separation. The multivalent interactions necessary for liquid-liquid phase separation have been extensively studied in vitro1,3. However, the regulation of this process in vivo is poorly understood. Here we identify an in vivo regulator of liquid-liquid phase separation through a genetic screen targeting factors required for Arabidopsis RNA-binding protein FCA function. FCA contains prion-like domains that phase-separate in vitro, and exhibits behaviour in vivo that is consistent with phase separation. The mutant screen identified a functional requirement for FLL2, a coiled-coil protein, in the formation of FCA nuclear bodies. FCA reduces transcriptional read-through by promoting proximal polyadenylation at many sites in the Arabidopsis genome3,4. FLL2 was required to promote this proximal polyadenylation, but not the binding of FCA to target RNA. Ectopic expression of FLL2 increased the size and number of FCA nuclear bodies. Crosslinking with formaldehyde captured in vivo interactions between FLL2, FCA and the polymerase and nuclease modules of the RNA 3'-end processing machinery. These 3' RNA-processing components colocalized with FCA in the nuclear bodies in vivo, which indicates that FCA nuclear bodies compartmentalize 3'-end processing factors to enhance polyadenylation at specific sites. Our findings show that coiled-coil proteins can promote liquid-liquid phase separation, which expands our understanding of the principles that govern the in vivo dynamics of liquid-like bodies.

Homotypic and heterotypic trans-assembly of human Rab-family small GTPases in reconstituted membrane tethering

Membrane tethering is a highly regulated event occurring during the initial physical contact between membrane-bounded transport carriers and their target subcellular membrane compartments, thereby ensuring the spatiotemporal specificity of intracellular membrane trafficking. Although Rab-family small GTPases and specific Rab-interacting effectors, such as coiled-coil tethering proteins and multisubunit tethering complexes, are known to be involved in membrane tethering, how these protein components directly act upon the tethering event remains enigmatic. Here, using a chemically defined reconstitution system, we investigated the molecular basis of membrane tethering by comprehensively and quantitatively evaluating the intrinsic capacities of 10 representative human Rab-family proteins (Rab1a, -3a, -4a, -5a, -6a, -7a, -9a, -11a, -27a, and -33b) to physically tether two distinct membranes via homotypic and heterotypic Rab-Rab assembly. All of the Rabs tested, except Rab27a, specifically caused homotypic membrane tethering at physiologically relevant Rab densities on membrane surfaces (e.g. Rab/lipid molar ratios of 1:100-1:3,000). Notably, endosomal Rab5a retained its intrinsic potency to drive efficient homotypic tethering even at concentrations below the Rab/lipid ratio of 1:3,000. Comprehensive reconstitution experiments further uncovered that heterotypic combinations of human Rab-family isoforms, including Rab1a/6a, Rab1a/9a, and Rab1a/33b, can directly and selectively mediate membrane tethering. Rab1a and Rab9a in particular synergistically triggered very rapid and efficient membrane tethering reactions through their heterotypic trans-assembly on two opposing membranes. In conclusion, our findings establish that, in the physiological context, homotypic and heterotypic trans-assemblies of Rab-family small GTPases can provide the essential molecular machinery necessary to drive membrane tethering in eukaryotic endomembrane systems.

Apical assemblies of intermediate filament-like protein FilP are highly dynamic and affect polar growth determinant DivIVA in Streptomyces venezuelae.

Streptomyces spp. grow as branching hyphae, building the cell wall in restricted zones at hyphal tips. The organization of this mode of polar growth involves three coiled-coil proteins: DivIVA and Scy, which form apical protein complexes referred to as polarisomes; and the intermediate filament-like protein FilP, which influences cell shape and interacts with both Scy and DivIVA. Here, we use live cell imaging of Streptomyces venezuelae to clarify the subcellular localization and dynamics of FilP and its effect on hyphal morphology. By monitoring a FilP-mCherry fusion protein, we show that FilP accumulates in gradient-like zones behind the hyphal tips. The apical gradient pattern of FilP localization is dependent on hyphal tip extension and immediately dissipates upon growth arrest. Fluorescence recovery after photobleaching experiments show that FilP gradients are dynamic and subject to subunit exchange during vegetative growth. Further, the localization of FilP at hyphal tips is not directly dependent on scy, even though the strongly perturbed morphology of most scy mutant hyphae is associated with mislocalization of FilP. Finally, we find that filP has an effect on the size and position of the foci of key polar growth determinant DivIVA. This effect likely contributes to the phenotype of filP mutants.