scholarly journals Two novel heteropolymer-forming proteins maintain multicellular shape of the cyanobacteriumAnabaenasp. PCC 7120

2019 ◽  
Author(s):  
Benjamin L. Springstein ◽  
Dennis J. Nürnberg ◽  
Christian Woehle ◽  
Julia Weissenbach ◽  
Marius L. Theune ◽  
...  
Keyword(s):  
Double Mutant ◽  
Protein Complexes ◽  
Coiled Coil ◽  
Filamentous Structure ◽  
Cellular Processes ◽  
Junction Protein ◽  
And Function ◽  
Pcc 7120

AbstractPolymerizing and filament-forming proteins are instrumental for numerous cellular processes such as cell division and growth. Their function in stabilization and localization of protein complexes and replicons is achieved by a filamentous structure. Known filamentous proteins assemble into homopolymers consisting of single subunits – e.g. MreB and FtsZ in bacteria – or heteropolymers that are composed of two subunits, e.g. keratin and α/β tubulin in eukaryotes. Here, we describe two novel coiled-coil-rich proteins (CCRPs) in the filament forming cyanobacteriumAnabaenasp. PCC 7120 (hereafterAnabaena) that assemble into a heteropolymer and function in the maintenance of theAnabaenamulticellular shape (termed trichome). The two CCRPs – Alr4504 and Alr4505 (named ZicK and ZacK) – are strictly interdependent for the assembly of protein filamentsin vivoand polymerize nucleotide-independentlyin vitro, similar to known intermediate filament (IF) proteins. A ΔzicKΔzacK double mutant is characterized by a zigzagged cell arrangement and hence a loss of the typical linearAnabaenatrichome shape. ZicK and ZacK interact with themselves, with each other, with the elongasome protein MreB, the septal junction protein SepJ and the divisome associate septal protein SepI. Our results suggest that ZicK and ZacK function in cooperation with SepJ and MreB to stabilize theAnabaenatrichome and are likely essential for the manifestation of the multicellular shape inAnabaena. Our study reveals the presence of filament-forming IF-like proteins whose function is achieved through the formation of heteropolymers in cyanobacteria.

scholarly journals Identification and characterization of novel filament-forming proteins in cyanobacteria

2019 ◽  
Author(s):  
Benjamin L. Springstein ◽  
Christian Woehle ◽  
Julia Weissenbach ◽  
Andreas O. Helbig ◽  
Tal Dagan ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Cytoskeletal Proteins ◽  
Tetratricopeptide Repeat ◽  
Cellular Processes ◽  
Tetratricopeptide Repeat Protein ◽  
Pcc 7120 ◽  
Pcc 7942

AbstractFilament-forming proteins in bacteria function in stabilization and localization of proteinaceous complexes and replicons; hence they are instrumental for myriad cellular processes such as cell division and growth. Here we present two novel filament-forming proteins in cyanobacteria. Surveying cyanobacterial genomes for coiled-coil-rich proteins (CCRPs) that are predicted as putative filament-forming proteins, we observed a higher proportion of CCRPs in filamentous cyanobacteria in comparison to unicellular cyanobacteria. Using our predictions, we identified nine protein families with putative intermediate filament (IF) properties. Polymerization assays revealed four proteins that formed polymers in vitro and three proteins that formed polymers in vivo. Fm7001 from Fischerella muscicola PCC 7414 polymerized in vitro and formed filaments in vivo in several organisms. Additionally, we identified a tetratricopeptide repeat protein - All4981 - in Anabaena sp. PCC 7120 that polymerized into filaments in vitro and in vivo. All4981 interacts with known cytoskeletal proteins and is indispensable for Anabaena viability. Although it did not form filaments in vitro, Syc2039 from Synechococcus elongatus PCC 7942 assembled into filaments in vivo and a Δsyc2039 mutant was characterized by an impaired cytokinesis. Our results expand the repertoire of known prokaryotic filament-forming CCRPs and demonstrate that cyanobacterial CCRPs are involved in cell morphology, motility, cytokinesis and colony integrity.

scholarly journals Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

2002 ◽  
Vol 159 (6) ◽  
pp. 993-1004 ◽  
Author(s):  
Christine L. Humphries ◽  
Heath I. Balcer ◽  
Jessica L. D'Agostino ◽  
Barbara Winsor ◽  
David G. Drubin ◽  
...  
Keyword(s):  
Binding Protein ◽  
Coiled Coil ◽  
Actin Binding ◽  
Complex Activity ◽  
Actin Binding Protein ◽  
Coiled Coil Domain ◽  
Allele Specific ◽  
And Function

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.

scholarly journals Watching cellular machinery in action, one molecule at a time

2016 ◽  
Vol 216 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Enrico Monachino ◽  
Lisanne M. Spenkelink ◽  
Antoine M. van Oijen
Keyword(s):  
Dynamic Behavior ◽  
Single Molecule ◽  
Protein Complexes ◽  
Imaging Techniques ◽  
Ensemble Averaging ◽  
Cellular Processes ◽  
Cellular Machinery

Single-molecule manipulation and imaging techniques have become important elements of the biologist’s toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.

scholarly journals Reconstitution of Two Recombinant LSm Protein Complexes Reveals Aspects of Their Architecture, Assembly, and Function

2005 ◽  
Vol 280 (16) ◽  
pp. 16066-16075 ◽  
Author(s):  
Bozidarka Zaric ◽  
Mohamed Chami ◽  
Hervé Rémigy ◽  
Andreas Engel ◽  
Kurt Ballmer-Hofer ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Cellular Transport ◽  
Core Domain ◽  
Assembly Pathway ◽  
Rna Targets ◽  
Transport Assay ◽  
Form Ring ◽  
And Function

Sm and Sm-like (LSm) proteins form complexes engaging in various RNA-processing events. Composition and architecture of the complexes determine their intracellular distribution, RNA targets, and function. We have reconstituted the human LSm1–7 and LSm2–8 complexes from their constituent componentsin vitro. Based on the assembly pathway of the canonical Sm core domain, we used heterodimeric and heterotrimeric sub-complexes to assemble LSm1–7 and LSm2–8. Isolated sub-complexes form ring-like higher order structures. LSm1–7 is assembled and stable in the absence of RNA. LSm1–7 forms ring-like structures very similar to LSm2–8 at the EM level. Ourin vitroreconstitution results illustrate likely features of the LSm complex assembly pathway. We prove the complexes to be functional both in an RNA bandshift and anin vivocellular transport assay.

scholarly journals Hyperglycemia downregulates Connexin36 in pancreatic islets via the upregulation of ICER-1/ICER-1γ

2013 ◽  
Vol 51 (1) ◽  
pp. 49-58 ◽  
Author(s):  
Jacques-Antoine Haefliger ◽  
Françoise Rohner-Jeanrenaud ◽  
Dorothée Caille ◽  
Anne Charollais ◽  
Paolo Meda ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Pancreatic Islets ◽  
Chronic Exposure ◽  
Prolonged Exposure ◽  
Mrna Levels ◽  
Adult Rats ◽  
Junction Protein ◽  
And Function

Channels formed by the gap junction protein Connexin36 (CX36) contribute to the proper control of insulin secretion. We previously demonstrated that chronic exposure to glucose decreases Cx36 levels in insulin-secreting cells in vitro. Here, we investigated whether hyperglycemia also regulates Cx36 in vivo. Using a model of continuous glucose infusion in adult rats, we showed that prolonged (24–48 h) hyperglycemia reduced the Cx36 gene Gjd2 mRNA levels in pancreatic islets. Accordingly, prolonged exposure to high glucose concentrations also reduced the expression and function of Cx36 in the rat insulin-producing INS-1E cell line. The glucose effect was blocked after inhibition of the cAMP/PKA pathway and was associated with an overexpression of the inducible cAMP early repressor ICER-1/ICER-1γ, which binds to a functional cAMP-response element in the promoter of the Cx36 gene Gjd2. The involvement of this repressor was further demonstrated using an antisense strategy of ICER-1 inhibition, which prevented glucose-induced downregulation of Cx36. The data indicate that chronic exposure to glucose alters the in vivo expression of Cx36 by the insulin-producing β-cells through ICER-1/ICER-1γ overexpression. This mechanism may contribute to the reduced glucose sensitivity and altered insulin secretion, which contribute to the pathophysiology of diabetes.

scholarly journals Structure and function of Spc42 coiled-coils in yeast centrosome assembly and duplication

2019 ◽  
Vol 30 (12) ◽  
pp. 1505-1522 ◽  
Author(s):  
Amanda C. Drennan ◽  
Shivaani Krishna ◽  
Mark A. Seeger ◽  
Michael P. Andreas ◽  
Jennifer M. Gardner ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Core Layer ◽  
Spindle Pole ◽  
Coiled Coils ◽  
Lipid Monolayer ◽  
Functional Roles ◽  
Spindle Pole Bodies ◽  
And Function

Centrosomes and spindle pole bodies (SPBs) are membraneless organelles whose duplication and assembly is necessary for bipolar mitotic spindle formation. The structural organization and functional roles of major proteins in these organelles can provide critical insights into cell division control. Spc42, a phosphoregulated protein with an N-terminal dimeric coiled-coil (DCC), assembles into a hexameric array at the budding yeast SPB core, where it functions as a scaffold for SPB assembly. Here, we present in vitro and in vivo data to elucidate the structural arrangement and biological roles of Spc42 elements. Crystal structures reveal details of two additional coiled-coils in Spc42: a central trimeric coiled-coil and a C-terminal antiparallel DCC. Contributions of the three Spc42 coiled-coils and adjacent undetermined regions to the formation of an ∼145 Å hexameric lattice in an in vitro lipid monolayer assay and to SPB duplication and assembly in vivo reveal structural and functional redundancy in Spc42 assembly. We propose an updated model that incorporates the inherent symmetry of these Spc42 elements into a lattice, and thereby establishes the observed sixfold symmetry. The implications of this model for the organization of the central SPB core layer are discussed.

scholarly journals Unraveling the mechanism of the cadherin-catenin-actin catch bond

2018 ◽  
Author(s):  
Shishir Adhikari ◽  
Jacob Moran ◽  
Christopher Weddle ◽  
Michael Hinczewski
Keyword(s):  
Protein Complexes ◽  
Optical Tweezer ◽  
Force Transducer ◽  
Salt Bridges ◽  
Lifetime Distributions ◽  
Catch Bond ◽  
The Individual ◽  
And Function

The adherens junctions between epithelial cells involve a protein complex formed by E-cadherin, β-catenin, α-catenin and F-actin. The stability of this complex was a puzzle for many years, since in vitro studies could reconstitute various stable subsets of the individual proteins, but never the entirety. The missing ingredient turned out to be mechanical tension: a recent experiment that applied physiological forces to the complex with an optical tweezer dramatically increased its lifetime, a phenomenon known as catch bonding. However, in the absence of a crystal structure for the full complex, the microscopic details of the catch bond mechanism remain mysterious. Building on structural clues that point to α-catenin as the force transducer, we present a quantitative theoretical model for how the catch bond arises, fully accounting for the experimental lifetime distributions. The model allows us to predict the energetic changes induced by tension at the interface between α-catenin and F-actin. It also identifies a significant energy barrier due to a network of salt bridges between two conformational states of β-catenin. By stabilizing one of these states, this barrier could play a role in how the complex responds to additional in vivo binding partners like vinculin. Since significant conformational energy barriers are a common feature of other adhesion systems that exhibit catch bonds, our model can be adapted into a general theoretical framework for integrating structure and function in a variety of force-regulated protein complexes.

scholarly journals The golgin protein Coy1 functions in intra-Golgi retrograde transport and interacts with the COG complex and Golgi SNAREs

2017 ◽  
Vol 28 (20) ◽  
pp. 2686-2700 ◽  
Author(s):  
Nadine S. Anderson ◽  
Indrani Mukherjee ◽  
Christine M. Bentivoglio ◽  
Charles Barlowe
Keyword(s):  
Protein Interactions ◽  
Coiled Coil ◽  
Retrograde Transport ◽  
Snare Proteins ◽  
Growth Defects ◽  
Cog Complex ◽  
Physical Interactions ◽  
And Function

Extended coiled-coil proteins of the golgin family play prominent roles in maintaining the structure and function of the Golgi complex. Here we further investigate the golgin protein Coy1 and document its function in retrograde transport between early Golgi compartments. Cells that lack Coy1 displayed a reduced half-life of the Och1 mannosyltransferase, an established cargo of intra-Golgi retrograde transport. Combining the coy1Δ mutation with deletions in other putative retrograde golgins (sgm1Δ and rud3Δ) caused strong glycosylation and growth defects and reduced membrane association of the conserved oligomeric Golgi (COG) complex. In contrast, overexpression of COY1 inhibited the growth of mutant strains deficient in fusion activity at the Golgi (sed5-1 and sly1-ts). To map Coy1 protein interactions, coimmunoprecipitation experiments revealed an association with the COG complex and with intra-Golgi SNARE proteins. These physical interactions are direct, as Coy1 was efficiently captured in vitro by Lobe A of the COG complex and the purified SNARE proteins Gos1, Sed5, and Sft1. Thus our genetic, in vivo, and biochemical data indicate a role for Coy1 in regulating COG complex-dependent fusion of retrograde-directed COPI vesicles.

scholarly journals Bridging the gap betweenin vitroandin vivoRNA folding

2016 ◽  
Vol 49 ◽  
Author(s):  
Kathleen A. Leamy ◽  
Sarah M. Assmann ◽  
David H. Mathews ◽  
Philip C. Bevilacqua
Keyword(s):  
Rna Structure ◽  
Rna Folding ◽  
Three Dimensional ◽  
Molecular Crowding ◽  
Cellular Processes ◽  
Cellular Environment ◽  
Folding Pathways ◽  
And Function

AbstractDeciphering the folding pathways and predicting the structures of complex three-dimensional biomolecules is central to elucidating biological function. RNA is single-stranded, which gives it the freedom to fold into complex secondary and tertiary structures. These structures endow RNA with the ability to perform complex chemistries and functions ranging from enzymatic activity to gene regulation. Given that RNA is involved in many essential cellular processes, it is critical to understand how it folds and functionsin vivo. Within the last few years, methods have been developed to probe RNA structuresin vivoand genome-wide. These studies reveal that RNA often adopts very different structuresin vivoandin vitro, and provide profound insights into RNA biology. Nonetheless, bothin vitroandin vivoapproaches have limitations: studies in the complex and uncontrolled cellular environment make it difficult to obtain insight into RNA folding pathways and thermodynamics, and studiesin vitrooften lack direct cellular relevance, leaving a gap in our knowledge of RNA foldingin vivo. This gap is being bridged by biophysical and mechanistic studies of RNA structure and function under conditions that mimic the cellular environment. To date, most artificial cytoplasms have used various polymers as molecular crowding agents and a series of small molecules as cosolutes. Studies under suchin vivo-likeconditions are yielding fresh insights, such as cooperative folding of functional RNAs and increased activity of ribozymes. These observations are accounted for in part by molecular crowding effects and interactions with other molecules. In this review, we report milestones in RNA foldingin vitroandin vivoand discuss ongoing experimental and computational efforts to bridge the gap between these two conditions in order to understand how RNA folds in the cell.

scholarly journals Chimeric 14-3-3 proteins for unravelling interactions with intrinsically disordered partners

2017 ◽  
Author(s):  
Nikolai N. Sluchanko ◽  
Kristina V. Tugaeva ◽  
Alfred A. Antson
Keyword(s):  
Structural Information ◽  
Protein Complexes ◽  
Cellular Processes ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
C Terminus ◽  
Transient Interactions ◽  
Partner Proteins

ABSTRACTIn eukaryotes, several proteins act as “hubs”, integrating signals from a variety of interacting partners that bind to the hub through intrinsically disordered regions. Not surprisingly, one of the major hubs, the 14-3-3 protein, that plays wide-ranging roles in cellular processes, has been linked with a number of disorders including neurodegenerative diseases and cancer. A partner protein usually binds with its phosphopeptide accommodated in an amphipathic groove (AG) of 14-3-3, a promising platform for therapeutic intervention. Protein plasticity in the groove allows to accommodate a range of phosphopeptides with different sequences. So far, in spite of mammoth effort, accurate structural information has been derived only for few 14-3-3 complexes with phosphopeptide-containing proteins or various short synthetic peptides. The progress has been prevented by intrinsic disorder of partner proteins and, in case of transient interactions, by the low affinity of phosphopeptides. We reasoned that these problems could be resolved by using chimeric 14-3-3 proteins with incorporated peptide sequences. We tested this hypothesis and found that such chimeric proteins are easy to design, express, purify and crystallize. We show that when attached to the C terminus of 14-3-3 via an optimal linker, peptides become stoichiometrically phosphorylated by protein kinase A during bacterial co-expression. We determined crystal structures for complexes of chimeric 14-3-3 protein fused with three different peptides. In most of the cases, the phosphopeptide is bound inside the AG, providing invaluable information on its interaction with the protein. This approach can reinvigorate studies of 14-3-3 protein complexes, including those with otherwise challenging low affinity phosphopeptides. Furthermore, 14-3-3-phosphopeptide chimeras can be useful for the design of novel biosensors for in vitro and in vivo imaging experiments.

Sign in / Sign up

Export Citation Format

Share Document