Cargo Complexes Research Articles

In situ structural analysis of the human nuclear pore complex.

SummaryNuclear pore complexes (NPCs) are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Elucidating their 110 MDa structure imposes a formidable challenge and requires in situ structural biology approaches. Fifteen out of about thirty nucleoporins (Nups) are structured and form the Y- and inner ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ∼60 nm in diameter 1. The scaffold is decorated with transport channel Nups that often contain phenylalanine (FG)-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y-complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here, we combined cryo electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modeling to generate the most comprehensive architectural model of the NPC to date. Our data suggest previously unknown protein interfaces across Y-complexes and to inner ring complex members. We demonstrate that the higher eukaryotic transport channel Nup358 (RanBP2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport channel Nups. We conclude that, similarly to coated vesicles, multiple copies of the same structural building block - although compositionally identical - engage in different local sets of interactions and conformations.

A non-canonical mechanism for Crm1-export cargo complex assembly.

The transport receptor Crm1 mediates the export of diverse cargos containing leucine-rich nuclear export signals (NESs) through complex formation with RanGTP. To ensure efficient cargo release in the cytoplasm, NESs have evolved to display low affinity for Crm1. However, mechanisms that overcome low affinity to assemble Crm1-export complexes in the nucleus remain poorly understood. In this study, we reveal a new type of RanGTP-binding protein, Slx9, which facilitates Crm1 recruitment to the 40S pre-ribosome-associated NES-containing adaptor Rio2. In vitro, Slx9 binds Rio2 and RanGTP, forming a complex. This complex directly loads Crm1, unveiling a non-canonical stepwise mechanism to assemble a Crm1-export complex. A mutation in Slx9 that impairs Crm1-export complex assembly inhibits 40S pre-ribosome export. Thus, Slx9 functions as a scaffold to optimally present RanGTP and the NES to Crm1, therefore, triggering 40S pre-ribosome export. This mechanism could represent one solution to the paradox of weak binding events underlying rapid Crm1-mediated export.

Development of techniques to optimize the technical parameters of transport cargo complexes

Transition to a competitive environment and open service market in the transport sector has high requirements for all aspects of its functioning. To ensure an adequate level of competitiveness, rail transport should provide the customers with the most efficient and low-cost services in both transportation, and cargo handling, which is impossible without efficient technical equipment of cargo complexes.As a result of the research, the dependences of the value of the admissible efficiency criterion - specific profit of TPC enterprises per unit of value of cargo complex fixed assets, on the amount of production resources - shunting locomotives, cargo handling machines and sizes of storage facilities were obtained.The results have shown the presence of a complex polynomial dependence of the operating costs of the shunting locomotives and mechanisms in the sphere of cargo operations on the number of service units. Optimum size of storage facilities stepwise depends on the car traffic volume intensity.Using the obtained dependences allows to optimize the technical equipment of existing cargo complexes and set their optimal process parameters at the design and equipping stage.

An Optimized Viral Delivery System to Expand the Genetic Code in Mammalian Cells

Co‐translational, site‐specific incorporation of unnatural amino acids (UAAs) by nonsense suppression has proven to be a powerful tool for investigating and engineering protein structure and functions. This methodology utilizes an engineered aminoacyl‐tRNA synthetase/tRNA (aaRS/tRNA) pair, orthogonal to the host cell, to deliver the UAA of interest in response to a nonsense codon. The ability to use this toolbox to investigate and manipulate protein function in human and other mammalian cells can be highly beneficial. The primary challenge is to efficiently co‐deliver all the genetic components, and a lack of understanding regarding optimal expression levels of these components. Viral vectors are ideal vehicles for delivery, potentially enabling application in a variety of mammalian cell lines, and even live tissues and animals.Using a unique viral delivery vector, and optimal expression of the genetic components, we have substantially enhanced UAA mutagenesis efficiency in mammalian cells. We identified the tRNA as the limiting component, while the traditional aaRS expression level was shown to be unfavorably high. The large and complex cargo could be efficiently packaged within our unique baculoviral delivery vector‐ showcasing the prowfess of this platform.

Axonal transport cargo motor count versus average transport velocity: is fast versus slow transport really single versus multiple motor transport?

Cargos have been observed exhibiting a "stop-and-go" behavior (i.e. cargo "pause"), and it has generally been assumed that these multi-second pauses can be attributed to equally long pauses of cargo-bound motors during motor procession. We contend that a careful examination of the isolated microtubule experimental record does not support motor pauses. Rather, we believe that the data suggests that motor cargo complexes encounter an obstruction that prevents procession, eventually detach and reattach, with this obstructed-detach-reattach sequence being observed in axon as a "pause." Based on this, along with our quantitative evidence-based contention that slow and fast axonal transport are actually single and multi-motor transport, we have developed a cargo level motor model capable of exhibiting the full range of slow to fast transport solely by changing the number of motors involved. This computational model derived using first-order kinetics is suitable for both kinesin and dynein and includes load-dependence as well as provision for motors encountering obstacles to procession. The model makes the following specific predictions: average distance from binding to obstruction is about 10 μm; average motor maximum velocity is at least 6 μm/s in axon; a minimum of 10 motors is required for the fastest fast transport while only one motor is required for slow transport; individual in-vivo cargo-attached motors may spend as little as 5% of their time processing along a microtubule with the remainder being spent either obstructed or unbound to a microtubule; and at least in the case of neurofilament transport, kinesin and dynein are largely not being in a "tug-of-war" competition.

Nup98 FG domains from diverse species spontaneously phase-separate into particles with nuclear pore-like permselectivity.

Nuclear pore complexes (NPCs) conduct massive transport mediated by shuttling nuclear transport receptors (NTRs), while keeping nuclear and cytoplasmic contents separated. The NPC barrier in Xenopus relies primarily on the intrinsically disordered FG domain of Nup98. We now observed that Nup98 FG domains of mammals, lancelets, insects, nematodes, fungi, plants, amoebas, ciliates, and excavates spontaneously and rapidly phase-separate from dilute (submicromolar) aqueous solutions into characteristic 'FG particles'. This required neither sophisticated experimental conditions nor auxiliary eukaryotic factors. Instead, it occurred already during FG domain expression in bacteria. All Nup98 FG phases rejected inert macromolecules and yet allowed far larger NTR cargo complexes to rapidly enter. They even recapitulated the observations that large cargo-domains counteract NPC passage of NTR⋅cargo complexes, while cargo shielding and increased NTR⋅cargo surface-ratios override this inhibition. Their exquisite NPC-typical sorting selectivity and strong intrinsic assembly propensity suggest that Nup98 FG phases can form in authentic NPCs and indeed account for the permeability properties of the pore.

Applications of CPPs in Genome Modulation of Plants.

Cell-penetrating peptides (CPPs) are a class of short peptides that are known to translocate inside living cells through the cell membrane. Many CPPs show an ability to bind and deliver macromolecular cargoes such as DNA, RNA and protein into living cells, making them excellent transfection and transduction agents with low cytotoxicity. While their use is well established in mammalian cell systems, they have also been explored in the last decade as transfection agents in plant cells. Their efficacy has been demonstrated in both monocot and dicot clades as well as a variety of tissues and cell cultures, from leaves to protoplasts. Factors affecting CPP and CPP-cargo uptake have been addressed with specific attention to the plant cell wall and classes of CPPs utilized in plant cell systems. It has been shown that internalization of most free peptides in plant cells has been dominated by direct translocation across the cell membrane, while CPP-macromolecular cargo complexes and conjugates were translocated via macropinocytosis. Moreover, functionalization of CPPs resulted in generation of peptides with specialized cargo delivery attributes, e.g., for specific subcellular targeting. Thus, the use of CPPs in plants presents a promising method for plant transgenesis as well as genome regulation and modification.

Extracellular vesicles from bone marrow mesenchymal stem/stromal cells transport tumor regulatory microRNA, proteins, and metabolites.

Human mesenchymal stem/stromal cells (hMSCs) have been shown to support breast cancer cell proliferation and metastasis, partly through their secretome. hMSCs have a remarkable ability to survive for long periods under stress, and their secretome is tumor supportive. In this study, we have characterized the cargo of extracellular vesicular (EV) fraction (that is in the size range of 40-150nm) of serum deprived hMSCs (SD-MSCs). Next Generation Sequencing assays were used to identify small RNA secreted in the EVs, which indicated presence of tumor supportive miRNA. Further assays demonstrated the role of miRNA-21 and 34a as tumor supportive miRNAs. Next, proteomic assays revealed the presence of ≈150 different proteins, most of which are known tumor supportive factors such as PDGFR-β, TIMP-1, and TIMP-2. Lipidomic assays verified presence of bioactive lipids such as sphingomyelin. Furthermore, metabolite assays identified the presence of lactic acid and glutamic acid in EVs. The co-injection xenograft assays using MCF-7 breast cancer cells demonstrated the tumor supportive function of these EVs. To our knowledge this is the first comprehensive -omics based study that characterized the complex cargo of extracellular vesicles secreted by hMSCs and their role in supporting breast cancers.

Distinct N-terminal regions of the exomer secretory vesicle cargo Chs3 regulate its trafficking itinerary.

Cells transport integral membrane proteins between organelles by sorting them into vesicles. Cargo adaptors act to recognize sorting signals in transmembrane cargos and to interact with coat complexes that aid in vesicle biogenesis. No coat proteins have yet been identified that generate secretory vesicles from the trans-Golgi network (TGN) to the plasma membrane, but the exomer complex has been identified as a cargo adaptor complex that mediates transport of several proteins in this pathway. Chs3, the most well-studied exomer cargo, cycles between the TGN and the plasma membrane in synchrony with the cell cycle, providing an opportunity to study regulation of proteins that cycle in response to signaling. Here we show that different segments of the Chs3 N-terminus mediate distinct trafficking steps. Residues 10–27, known to mediate retention, also appear to play a role in internalization. Residues 28–52 are involved in transport to the plasma membrane and recycling out of endosomes to prevent degradation in the vacuole. We also present the crystal structure of residues 10–27 bound to the exomer complex, suggesting different cargo adaptors could compete for binding to this segment, providing a potential mechanism for regulation.

Strategic management of transport cargo complex

Purpose. Making the qualitative administrative decisions defining strategy and tactics of transport cargo complexes development, and also its subsystems, is possible only in the presence of flexible optimization model. This model has to consider multiparametricity and multicriteriality of the given task, uncertainty and vagueness of input information, and also to provide process automation of searching the best parameters of the given production facility. The purpose of the research is to develop procedures for the strategic management of complex with view of the most important factors and their stochastic nature, which will execute the improvement of technical equipment of TCC. Methodology. The problem of strategic management is based on solving the complex of issues of the optimal number of shunting locomotives, optimal processing capability of handling the front and rational capacity of warehouses. The problem is solved on the basis of the proposed optimality criterion – the specific set of profit per unit of capital assets of freight industry. The listed problems are solved using simulation modeling of the freight industry. Findings. The use of developed procedure allows one to improve the technical equipment of the freight stations and complexes. Originality. For the first time it was developed the procedure of strategic management of development. This procedure allows taking into account the probabilistic nature of demand for services of transport freight complexes and technological processes of client services on the complex stations. The proposed procedure can be applied during when planning the investments in the creation of transport freight complexes. Practical value. Use as a basic tool of simulation models of complex cargo operation allows estimating the effectiveness of the capital investments, the level of operating costs, as well as the quality of meeting the demands of potential customers in transportations at the stage of transport cargo complex.

Effects of cargo molecules on membrane perturbation caused by transportan10 based cell-penetrating peptides

Cell-penetrating peptides with the ability to escape endosomes and reach the target are of great value as delivery vectors for different bioactive cargoes and future treatment of human diseases. We have studied two such peptides, NickFect1 and NickFect51, both originated from stearylated transportan10 (PF3). To obtain more insight into the mechanism(s) of peptide delivery and the biophysical properties of an efficient vector system, we investigated the effect of different bioactive oligonucleotide cargoes on peptide–membrane perturbation and peptide structural induction. We studied the membrane interactions of the peptides with large unilamellar vesicles and compared their effects with parent peptides transportan10 and PF3. In addition, cellular uptake and peptide-mediated oligonucleotide delivery were analyzed. Calcein leakage experiments showed that similar to transportan10, NickFect51 caused a significant degree of membrane leakage, whereas NickFect1, similar to PF3, was less membrane perturbing. The results are in agreement with previously published results indicating that NickFect51 is a more efficient endosomal escaper. However, the presence of a large cargo like plasmid DNA inhibited NickFect's membrane perturbation and cellular uptake efficiency of the peptide was reduced. We conclude that the pathway for cellular uptake of peptide complexes is cargo dependent, whereas the endosomal escape efficacy depends on peptide hydrophobicity and chemical structure. For small interfering RNA delivery, NickFect51 appears to be optimal. The biophysical signature shows that the peptide alone causes membrane perturbation, but the cargo complex does not. These two biophysical characteristics of the peptide and its cargo complex may be the signature of an efficient delivery vector system.

Myelination and Oligodendrocyte Functions in Psychiatric Diseases

Myelination and Oligodendrocyte Functions in Psychiatric Diseases

Navigation and motion control in space debris removal

The article proposes a system for removing space debris from the geostationary orbit (GEO). The system is based on advanced space solutions and rational scheme of collecting space debris by reusable automatic long-use space vehicles. Creation of such a system will probably encourage a number of innovative space technologies such as reactive motion with high specific impulse, interorbital transfers and space cargo transportation, automatic control and complex operations performed by automated systems.

Transport workers and global supply chain governance

INTERNATIONAL union rights Page 22 Volume 21 Issue 4 2014 REPORT ❐ LABOUR RIGHTS AND THE SUPPLY CHAIN Garments produced in the Indian state of Gujarat are likely to be handled by at least 13 different groups of transport workers before arriving at a UK retail store lower cost countries in the late 1940s. By registering vessels in countries such as Panama and Liberia, national regulations could be evaded. Over the past six decades ITF unions have fought back. Starting with industrial action by dockers against ships with poor working conditions, the FOC campaign has resulted in an international collective bargaining system that covers one quarter of all FOC vessels. The ITF has also succeeded in entrenching its influence in international law, such as through the ILO Maritime Labour Convention, which requires Port States to enforce minimum labour regulations. For all the historical achievements of the ITF’s FOC campaign, the downward pressure on the labour standards of transport workers is widespread . On a macro scale, the rapid growth of global trade has moved many transport jobs into nonunion spaces. A third of global shipping container volumes are now handled in Chinese ports. The much smaller statelet of Dubai, meanwhile, has carved out a niche as a global transhipment hub with its major port, land logistics, and air cargo complex. On a micro scale, new transport infrastructure has often been established right alongside traditional bastions of union power. During the mid2000s , the historically well-organised port of Mumbai was effectively wound down when new container ports were constructed a short distance down the coast. Union members that sought recognition in one these ports were the subject of repeated physical violence and intimidation. Halfway round the world in California, the immense but almost entirely non-union Inland Empire logistics complex, which employs 90,000 workers, has emerged next door to some of the world’s most highly organised dock workers. Even where transport workers have remained unionised, the relentless pressure on cost structures from lead firms has put them under enormous pressure. In many countries, low barriers to entry in the trucking sector help to create intense competition and often dangerous pressure on working conditions. In Australia, for example, trucking was the cause of 30 percent of all occupational deaths over a 10 period, and truck drivers specifically are 16 times more likely to be killed on the job than the average worker. The issues facing transport workers, therefore, may not be as high profile as the issues facing production workers. Nevertheless, there is still a serious governance gap, and a necessity to develop robust frameworks that hold lead firms accountable for labour standards in their transport operations. I t is widely accepted that lead firms have a major responsibility to govern labour standards in global supply chains. A range of regulation initiatives such as the OECD Guidelines on Multinational Enterprises, the UN Global Compact, and the Bangladesh Accord on Building and Fire Safety, identify the ultimate responsibilities of lead firms with varying degrees of precision. Production workers have been the major focus of these initiatives. Whether it is Indian garment workers making goods for European retailers, or Colombian plantation workers harvesting flowers for North American supermarkets, labour standards at the production end has been the main point of scrutiny. For many observers, it is here that the most serious labour rights abuses and governance failures occur. Comparatively little attention has been paid to the role of transport workers. And yet, the involvement of transport workers in these chains is widespread, and the governance arrangements for these workers is highly uneven. Take the example of the garment supply chain from north west India to northern Europe. Garments produced in the Indian state of Gujarat are likely to be handled by at least 13 different groups of transport workers across four different modes before arriving at a UK retail store. The journey of a typical consignment of shirts would look roughly like this. Arriving by truck in the port of Mundra’s logistics zone, they will first be stuffed into containers. The shirts would then be loaded by dockers onto a container ship heading for Dubai. Here, the shirts will be unloaded and...

Structural Characterisation of the Nuclear Import Receptor Importin Alpha in Complex with the Bipartite NLS of Prp20

The translocation of macromolecules into the nucleus is a fundamental eukaryotic process, regulating gene expression, cell division and differentiation, but which is impaired in a range of significant diseases including cancer and viral infection. The import of proteins into the nucleus is generally initiated by a specific, high affinity interaction between nuclear localisation signals (NLSs) and nuclear import receptors in the cytoplasm, and terminated through the disassembly of these complexes in the nucleus. For classical NLSs (cNLSs), this import is mediated by the importin-α (IMPα) adaptor protein, which in turn binds to IMPβ to mediate translocation of nuclear cargo across the nuclear envelope. The interaction and disassembly of import receptor:cargo complexes is reliant on the differential localisation of nucleotide bound Ran across the envelope, maintained in its low affinity, GDP-bound form in the cytoplasm, and its high affinity, GTP-bound form in the nucleus. This in turn is maintained by the differential localisation of Ran regulating proteins, with RanGAP in the cytoplasm maintaining Ran in its GDP-bound form, and RanGEF (Prp20 in yeast) in the nucleus maintaining Ran in its GTP-bound form. Here, we describe the 2.1 Å resolution x-ray crystal structure of IMPα in complex with the NLS of Prp20. We observe 1,091 Å2 of buried surface area mediated by an extensive array of contacts involving residues on armadillo repeats 2-7, utilising both the major and minor NLS binding sites of IMPα to contact bipartite NLS clusters 17RAKKMSK23 and 3KR4, respectively. One notable feature of the major site is the insertion of Prp20NLS Ala18 between the P0 and P1 NLS sites, noted in only a few classical bipartite NLSs. This study provides a detailed account of the binding mechanism enabling Prp20 interaction with the nuclear import receptor, and additional new information for the interaction between IMPα and cargo.

Redox-regulated Cargo Binding and Release by the Peroxisomal Targeting Signal Receptor, Pex5

In its role as a mobile receptor for peroxisomal matrix cargo containing a peroxisomal targeting signal called PTS1, the protein Pex5 shuttles between the cytosol and the peroxisome lumen. Pex5 binds PTS1 proteins in the cytosol via its C-terminal tetratricopeptide domains and delivers them to the peroxisome lumen, where the receptor·cargo complex dissociates. The cargo-free receptor is exported to the cytosol for another round of import. How cargo release and receptor recycling are regulated is poorly understood. We found that Pex5 functions as a dimer/oligomer and that its protein interactions with itself (homo-oligomeric) and with Pex8 (hetero-oligomeric) control the binding and release of cargo proteins. These interactions are controlled by a redox-sensitive amino acid, cysteine 10 of Pex5, which is essential for the formation of disulfide bond-linked Pex5 forms, for high affinity cargo binding, and for receptor recycling. Disulfide bond-linked Pex5 showed the highest affinity for PTS1 cargo. Upon reduction of the disulfide bond by dithiothreitol, Pex5 transitioned to a noncovalent dimer, concomitant with the partial release of PTS1 cargo. Additionally, dissipation of the redox balance between the cytosol and the peroxisome lumen caused an import defect. A hetero-oligomeric interaction between the N-terminal domain (amino acids 1-110) of Pex5 and a conserved motif at the C terminus of Pex8 further facilitates cargo release, but only under reducing conditions. This interaction is also important for the release of PTS1 proteins. We suggest a redox-regulated model for Pex5 function during the peroxisomal matrix protein import cycle.

The interplay between RPGR, PDEδ and Arl2/3 regulate the ciliary targeting of farnesylated cargo

Defects in primary cilia result in human diseases known as ciliopathies. The retinitis pigmentosa GTPase regulator (RPGR), mutated in the most severe form of the eye disease, is located at the transition zone of the ciliary organelle. The RPGR-interacting partner PDEδ is involved in trafficking of farnesylated ciliary cargo, but the significance of this interaction is unknown. The crystal structure of the propeller domain of RPGR shows the location of patient mutations and how they perturb the structure. The RPGR·PDEδ complex structure shows PDEδ on a highly conserved surface patch of RPGR. Biochemical experiments and structural considerations show that RPGR can bind with high affinity to cargo-loaded PDEδ and exposes the Arl2/Arl3-binding site on PDEδ. On the basis of these results, we propose a model where RPGR is acting as a scaffold protein recruiting cargo-loaded PDEδ and Arl3 to release lipidated cargo into cilia.

Structural Basis for Cell-Cycle-Dependent Nuclear Import Mediated by the Karyopherin Kap121p

Kap121p (also known as Pse1p) is an essential karyopherin that mediates nuclear import of a plethora of cargoes including cell cycle regulators, transcription factors, and ribosomal proteins in Saccharomyces cerevisiae. It has been proposed that the spindle assembly checkpoint signaling triggers molecular rearrangements of nuclear pore complexes and thereby arrests Kap121p-mediated nuclear import at metaphase, while leaving import mediated by other karyopherins unaffected. The Kap121p-specific import inhibition is required for normal progression through mitosis. To understand the structural basis for Kap121p-mediated nuclear import and its unique regulatory mechanism during mitosis, we determined crystal structures of Kap121p in isolation and also in complex with either its import cargoes or nucleoporin Nup53p or RanGTP. Kap121p has a superhelical structure composed of 24 HEAT repeats. The structures of Kap121p–cargo complexes define a non-conventional nuclear localization signal (NLS) that has a consensus sequence of KV/IxKx1-2K/H/R. The structure of Kap121p–Nup53p complex shows that cargo and Nup53p compete for the same high-affinity binding site, explaining how Nup53p binding forces cargo release when the Kap121p-binding site of Nup53p is exposed during mitosis. Comparison of the NLS and RanGTP complexes reveals that RanGTP binding not only occludes the cargo-binding site but also forces Kap121p into a conformation that is incompatible with NLS recognition.

Don’t get stuck in the pore

EMBO J (2013) 32 2, 204–218 doi:10.1038/emboj.2012.302; published online November302012 Eukaryotic cells critically rely on a constant and intensive exchange of macromolecules between the cytoplasm and nucleoplasm across the barrier of the nuclear envelope. Nuclear pore complexes (NPCs) function as gates to mediate this transport, but why they grant the selective passage of transport receptor–cargo complexes and at the same time exclude most other cellular macromolecules is incompletely understood. In this issue of The EMBO Journal, Labokha et al (2012) extend our view on how certain NPC proteins form a sieve-like meshwork within the pore that is crucial for these functions.

Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes

Nuclear pore complexes (NPCs) control the traffic between cell nucleus and cytoplasm. While facilitating translocation of nuclear transport receptors (NTRs) and NTR·cargo complexes, they suppress passive passage of macromolecules ⩾30 kDa. Previously, we reconstituted the NPC barrier as hydrogels comprising S. cerevisiae FG domains. We now studied FG domains from 10 Xenopus nucleoporins and found that all of them form hydrogels. Related domains with low FG motif density also substantially contribute to the NPC's hydrogel mass. We characterized all these hydrogels and observed the strictest sieving effect for the Nup98-derived hydrogel. It fully blocks entry of GFP-sized inert objects, permits facilitated entry of the small NTR NTF2, but arrests importin β-type NTRs at its surface. O-GlcNAc modification of the Nup98 FG domain prevented this arrest and allowed also large NTR·cargo complexes to enter. Solid-state NMR spectroscopy revealed that the O-GlcNAc-modified Nup98 gel lacks amyloid-like β-structures that dominate the rigid regions in the S. cerevisiae Nsp1 FG hydrogel. This suggests that FG hydrogels can assemble through different structural principles and yet acquire the same NPC-like permeability.