34 research outputs found
Kap-Centric control of nuclear pores based on promiscuous binding to FG nucleoporins
Get PDFNuclear pore complexes (NPCs) are remarkable molecular machines that perforate the nuclear envelope (NE) in eukaryotic cells and mediate the rapid bidirectional traffic of hundreds of proteins, ribonucleoproteins, and metabolites across the nuclear envelope. Their enormous structure is composed of multiple copies of 30 different proteins (Nups) that add up to 60 – 120 MDa of mass depending on the organism. Each NPC contains a 50 nm-diameter central channel through which only molecules smaller than ~40 kDa or ~5 nm in size can diffuse passively. The movement of larger molecules is impaired by a permeability barrier generated by ~200 partly intrinsically disordered phenylalanine-glycine (FG)-rich nucleoporins (FG Nups) that are tethered to the NPC transport channel surface. These FG Nups interact promiscuously with nuclear transport receptors (NTRs), such as karyopherins (Kaps; e.g. Kap-beta1) or NTF2, that mediate rapid trafficking of cargoes.
Given that the number of FG repeats per FG Nup also varies from 5 to ~50, NTR-FG Nup binding involves highly multivalent interactions, which are generally known to impart a strong avidity that enhances stability and specificity. However, this is paradoxical in the context of the NPC, because the high submicromolar Kap-beta1-FG domain binding affinities predict slow off rates (given a diffusion-limited on rate) that contradict the rapid (~5 ms) in vivo dwell time. As this implies, Kap-FG binding ought to be sufficiently strong to ensure selectivity but also weak enough to promote fast translocation through the NPC. Nonetheless, an explanation as to how promiscuous binding of FG Nups to NTRs is balanced against the mechanistic control of the FG domain barrier is still lacking.
The purpose of my work was to investigate FG Nup-NTR binding promiscuity and multivalency by measuring the interaction kinetics, binding affinity and in situ associated conformational changes in Nsp1p FG domains when binding NTF2 and Kap-beta1, both separately and together. Experimentally, this was achieved by using a novel surface plasmon resonance technique to correlate in situ mechanistic changes (molecular occupancy and conformational changes) with FG Nup-NTR binding.
The obtained results show that surface-tethered Nsp1p FG domains form molecular brushes at physiological conditions. Kap-beta1 binding provokes brush extension while partitioning into a fast and slow kinetic phase, where the latter may form an integral part of the FG domain barrier. In contrast, NTF2 binding to pristine Nsp1p layers induced collapse, but not under competing interactions from Kap-beta1. Therefore, promiscuous binding of NTF2 to Kap-beta1-preloaded Nsp1p attenuates NTF2 towards higher off rates and more transient interactions.
My work demonstrates that promiscuous binding of NTRs to FG Nups ought to influence nucleocytoplasmic transport. This depends on the concentration, size and binding strength of each NTR. Indeed, some form of hierarchy may exist between different NTRs such that their relative concentrations may impact NPC barrier function. This interpretation departs from the conventional view that the FG Nups alone form the NPC permeability barrier. Rather I conclude that concentrating NTRs in the NPC transport channel also contributes to generating crowding-based selective barrier function of the pore
Author Correction: Cancer-associated fibroblasts induce metalloprotease-independent cancer cell invasion of the basement membrane
No full textIn the original version of this Article, financial support and contributions in manuscript preparation were not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include the following:‘M.P. and P.O. would like to thank Prof. Roderick Y.H. Lim for advice during manuscript preparation and for providing the laboratory and microscopy infrastructure.… [We also thank] the NanoteraProject, awarded to the PATLiSciII Consortium (M.P and P.O)…’</jats:p
Barrier Dynamics of Nuclear Pore Complexes and Biomimetic Nanopores
Get PDFNuclear pore complexes (NPCs) mediate macromolecular traffic between the cytoplasm and the nucleus in eukaryotic cells. Tethered within each ~60 nm-diameter NPC lie numerous intrinsically disordered proteins that bear phenylalanine-glycine (FG) repeats known as FG nucleoporins (FG Nups). The FG Nups establish a selective barrier that impedes the passage of non-specific cargoes but rapidly yields to cargo-carrying transport receptors. However, the basic functional form of the FG Nups remains unresolved with respect to their spatiotemporal behaviour inside native NPCs. Here, we use high-speed atomic force microscopy (HS-AFM) to visualize nanoscopic FG Nup behaviour inside Xenopus laevis oocyte NPCs at near transport-relevant timescales. Our results show that the NPC channel is circumscribed by highly flexible, dynamically fluctuating FG Nups that elongate and retract in a stochastic manner consistent with the diffusive motion of tethered polypeptide chains. On this basis, extended FG Nups can momentarily interlink or coalesce into short-lived metastable condensates in the central channel, but do not cohere into a static meshwork that spans the entire pore. By resolving the time-dependent behaviour of FG Nups in the NPC, our findings bring consensus to barrier models that mainly disagree on static interpretations of how the FG Nups are spatially arranged in the pore. Furthermore, HS-AFM has been used to study the behavior of polyethylene glycol (PEG) polymer chains tethered inside of artificial nanopores. Our data shows that longer PEG chains serve are more effective in forming a barrier in pore than short PEG polymers. This serves as a strategy to design bio-mimetic nanopores with NPC-like functionality in the future
Towards solid-state biosensors for evidence-based management of febrile illness
Get PDFFever is a major reason for seeking medical care globally. It is the most prevalent symptom of infection, whether viral, bacterial, parasitic or fungal. The unspecific nature of fever makes it particularly challenging to identify a specific etiology, especially in low- and middle-income countries where diagnostic resources are scarce or absent. Incorrect or delayed diagnosis of acute fever is causing many preventable deaths and fuels the global rise in antimicrobial resistance. There is an urgent need for a simple to use, inexpensive and rapid diagnostic testing technology which can detect a multiplexed panel of biomarkers relevant to diagnosing and managing febrile illness. In this thesis, we provide a first approach based on solid-state microfabricated electrochemical sensors that are well suited for mass-manufacturing at low costs. We first report about a pH sensor based on a single layer of conductive TiN, which greatly simplifies pH sensor fabrication. Then, we demonstrate the sensing of dissolved oxygen using a nanotitration approach which does not require an oxygen-permeable membrane. We further demonstrate that our approach yields additional information on the buffer capacity of a solution. Next, we introduce the idea of redox-active nanopores for sensing proteins. We show that the redox-cycling current in such pores exhibits a weaker dependence on the pore diameter than the ionic current used in traditional nanopore analytics. This is advantageous for an increased signal-to-noise ratio and ultimately for improving the accessibility of nanopore analytics on proteins. Finally, we discuss how further development of these sensors could enable the detection of hypoxemia, anemia and various host protein markers to differentiate between bacterial and viral infections and significantly reduce antibiotics prescription among febrile patients while improving treatment outcome
Rebuilding Nuclear Pore Complex In Vitro
Get PDFThe hallmark of a eukaryotic cell is the segregation of genetic material within the cell’s nucleus. The nucleus is separated from the cytoplasm by a double lipid bilayer termed nuclear envelope (NE) that contains a number of proteinaceous channels known as nuclear pore complexes (NPCs).
NPCs enable molecular transport between the nucleus and cytoplasm, termed nucleocytoplasmic transport (NCT), which is bidirectional, fast and selective. Three major groups of proteins are continuously trafficking across the NPC’s central channel to orchestrate NCT. These are transport receptors, known as karyopherins (Kaps), signal- specific cargoes and a small GTPase (i.e., Ran) that sustains the process.
Interestingly, NPCs restrict or promote cargo translocation via biochemical selectivity and not size exclusion per se. Recent findings suggest that the NPC transport selective barrier is regulated by Kaps. This so-called Kap-centric regulation considers Kaps as integral constituents of the NPC that reinforce its selective barrier against large nonspecific macromolecules while simultaneously promoting the transport of specific cargoes. Nonetheless, an understanding of how Kaps contribute to NPC function remains incomplete.
The aim of this study was to rebuild NPC function in vitro.
First, we investigated how Kaps might reinforce the NPC transport barrier to establish a gradient of Ran guanosine triphosphate (RanGTP) and Ran guanosine diphosphate (RanGDP) in the nucleus and cytoplasm, respectively. Here, we show that the binding of RanGTP to Kaps at the NPC prevents its leakage into the cytosol.
Next, we show that two NPC pore membrane proteins are able to self-assemble into 20 nm- diameter nanopores following their reconstitution into lipid bilayers. This work represents a key step toward using nanopores as a de novo platform to construct additional NPC mimics with the aim of promoting NCT-like selective transport
Development and ssage of micro- and nanofluidic devices for nanoparticle trapping, sorting and biosensing
Get PDFMicrofluidics has revolutionized life sciences by introducing the tools to perform complex scientific studies in a simpler yet robust and reliable way. Miniaturization of bench-top processing tools using micro- and nanofluidic devices enables handling biological samples in a physiologically relevant environment to execute complex studies that were not possible before. Organ on a chip, lab on a chip, point-of-care diagnosis, biosensing, miniaturized PCR tools, etc., are some of the previously inconceivable examples in a portable device form. Due to the scale of the device dimensions in such microfluidic devices, small volume handling and processing have become noticeably effortless.
Among various applications of micro- and nanofluidic devices, molecular sensing, nanoparticle separation, sorting, trapping, and processing are of significant impact due to their feasibility of implementation in most of the fluidic devices. Single-particle trapping is an effective approach to study the fundamental properties of molecules in their physiological environment. Various active and passive methods exist to execute single-particle studies, such as optical tweezers, magnetic tweezers, dielectrophoretic trapping, hydrodynamic trapping, geometrical trapping, and electrostatic trapping. In the case of active methods, such as optical and magnetic tweezers, precise control of molecular motion is possible at the cost of a complex setup with external force sources. However, high-throughput single-particle trapping and manipulation are not feasible in a way that can be achieved using passive methods such as geometry induced electrostatic (GIE) trapping and geometrical trapping.
This thesis focuses on developing integrated micro and nanofluidic devices for 1) high throughput contact free electrostatic trapping of single nanoparticles and 2) size based nanoparticle separation, sorting, and trapping for biosensing applications. The high-throughput single-particle trapping was achieved by developing fluidic devices utilizing the GIE trapping. A GIE trapping fluidic device comprises nanochannels embedded with nanostructures, such as slits, cylinders, and grids. These nanostructures enable the formation of electrostatic potential traps inside the nanoindentations, forcing negatively charged nano objects to attain a position inside them to minimize their self-energy. In conventional GIE trapping devices, negatively charged molecules, such as DNA, viruses, and gold nanoparticles (Au NPs), can be easily trapped in the electrostatic traps.
This thesis presents the development and fabrication of GIE trapping devices using 1) glass substrate and 2) polydimethylsiloxane (PDMS) polymer. These substrates attain a net negative surface charge density in an aqueous solution (pH > 2) due to the self-dissociation of terminal silanol groups. Therefore, glass and PDMS based fluidic devices are only usable for the confinement of negatively charged nano objects. In this work, the scope of these fluidic devices was extended to the trapping of positively charged nano objects by using surface modification methods for both glass and PDMS based fluidic devices. The surface modification of glass‑based nanofluidic devices was achieved by modifying the inside of the GIE-trapping device by the adsorption of a single layer of polyelectrolyte (poly(ethyleneimine), PEI). The PEI layer modifies the negatively charged glass surface to a positively charged surface and allows for the trapping of positively charged nanoparticles. However, the surface modifying procedure for the glass based GIE trapping device was demanding and required 4 to 5 days. To have an efficient surface modification process, PDMS based GIE trapping devices were introduced.
The introduction of PDMS based fluidic devices for positively charged nano objects has improved the throughput for device fabrication and surface modification. Furthermore, two polyelectrolyte layers (1: poly(ethyleneimine) and 2: poly(styrenesulfonate)) deposition is presented in this work using PDMS based devices to demonstrate the possibility of achieving homogeneously charged surface using multi-polyelectrolyte layers. The efficiency of these devices with surface charge reversal was comparable to native GIE trapping devices, demonstrating the successful and homogeneous surface modification.
The trapping efficiency and device performance of a GIE Trapping device rely on the geometry of the device and the interaction between the charged particle and the device surface. Therefore, extensive optimization of the device geometry is essential to achieve efficient GIE trapping in a fluidic device. In this work, two different approaches, 1) charged particle inclusive simulation and 2) point charge approximation simulation, are presented to optimize the geometrical parameters of a GIE trapping device numerically. To compare numerical results with experimental data, a cylindrical nanopocket design was used to represent a nanotrap to confine a charged gold nanoparticle.
The charged-particle inclusive simulations are demanding, but provide more accurate results for attainable particle stiffness constant using crucial geometrical parameters of the device, size and charge of the particle of interest, and the salt concentration of the solution. Comparatively, point-charge approximation simulations are faster and give appropriate results of particle trapping stiffness constant, residence time, etc. Here, point-charge approximation simulations are used for efficiently identifying the trends of trapping strength of a device based on critical geometrical parameters, i.e., the height of the nanochannel and the nanopocket and the diameter of the nanopocket. The point charge approximation simulations demonstrated that the trapping strength of a particle inside a nanotrap could be enhanced by increasing the trap height or reducing the channel height. Additionally, the trapping strength of a nanotrap can be modified by changing the diameter of the nanopocket; however, reduction or enlargement of the pocket diameter from the optimum diameter reduces the trapping strength of the nanotrap. For effective GIE trapping, it is important to use a solution with low ionic or salt concentration ( 10-4 pN/nm) in order to avoid screening of the electrostatic field from the charged device surface. A detailed comparison of both approaches, numerical calculations, and experimental results are presented, demonstrating their advantages and disadvantages.
While there are many advantages of GIE trapping devices for molecular trapping, one major disadvantage is the reduced functionality of the devices for body fluids that contain high salt concentrations. Due to the high ionic concentration in the body fluids, the electrostatic effect of the charged device surface gets screened, leading to no potential trap for the confinement of charged nano objects. Therefore, a new design of the fluidic device is developed for biosensing applications that can use body fluids to extract the target molecules for molecular sensing. The fluidic device exploited geometrical sieving, deterministic lateral displacement (DLD) arrays, and geometrical trapping for particle separation, sorting, and trapping, respectively. The separation of unwanted macro- and micro-particles was achieved in the separation chamber, followed by the size sorting of target molecule adsorbed nanoparticles and, later, the size based trapping of these nanoparticles in the detection area. The motion of the solution and nanoparticle throughout the device was observed using interferometric scattering detection (iSCAT) microscopy, whereas, for molecular sensing, Raman spectroscopy was used at the detection area to achieve a few pg/ml detection limit. The device has the potential for applications in early multi disease diagnosis for diseases that can be detected using antigen-antibody complex formation on antibody-coated nanoparticles.
The presented GIE trapping devices can be used to achieve high-throughput single-nanoparticle trapping, whereas geometrical particle trapping devices can be used to perform size-selective nanoparticle trapping for molecular sensing. Both methods are effective for studies conducted in an aqueous environment and have the potential to be used in molecular studies, disease diagnosis, biological studies, etc., for research and commercial purposes. Demonstrated device fabrication methods and surface modification procedures allow improved productivity and yield of the GIE trapping devices. The device geometry of a GIE trapping device can be optimized further using the presented numerical calculations. Therefore, the work presented here advances the research in the field of GIE trapping and geometrical trapping and opens up new possibilities for both basic and applied research in several fields, such as biophysics, molecular dynamics, diagnostics, and molecular detection
Auxiliary barrier function of karyopherins at the Nuclear Pore Complex
Get PDFNuclear pore complexes (NPCs) are highly selective gateways that mediate nucleocytoplasmic transport (NCT) in eukaryotic cells. Recent discoveries have shown that leaky NPCs and defective NCT are linked to aging, neurodegenerative disorders, and viral pathogenesis. Nevertheless, their exact underlying cause(s) are unknown, reflecting an incomplete understanding of the key regulatory aspects of NPC function. At the heart of this problem lies the NPC permeability barrier, whose behavior has been largely modeled after the in vitro behavior of intrinsically disordered proteins termed phenylalanine-glycine nucleoporins (FG Nups). Nonetheless, this view is puzzling since certain key soluble nuclear transport receptors called β-karyopherins (Kapβs) are strongly enriched within NPCs in vivo.
The experimental results reported in this thesis show that two major Kapβs, Kapβ1 (importinβ) and CRM1 (exportin1) are essential for fortifying the NPC permeability barrier against defective NCT and nuclear leakage in vivo. A further surprise is that CRM1 partially compensates for Kapβ1 upon depletion of the latter from the NPC, which suggests that Kapβ1 and CRM1 engage in a balancing act to reinforce NPC barrier function. Combining ex vivo and biophysical experimentation, as well as computational modeling, we further show how the occupancy of different Kapβs at the NPC is constrained by their size, cellular abundance, binding avidity to the FG Nups, and competition with other Kapβs, such as demonstrated for another Kapβ, Importin-5 (Imp5). Taken together, these findings provide important intersection points and raise new questions with respect to the causes of NPC leakage and defective NCT in aging and cellular pathologies
Probing and engineering mechanostable protein complexes
Get PDFThe mechanostability of proteins plays an important role in various biological processes, for example cell adhesion and pathogen invasion. Single-molecule force spectroscopy (SMFS) is a powerful tool to understand the molecular mechanisms of mechanostable proteins, gain a mechanistic insight into biological systems and also direct the engineering of biomolecules for desirable mechanical properties, for example enhanced mechanostability.
One family of highly mechanostable cell adhesion proteins is the dockerin (Doc)-cohesin (Coh) family from cellulosomes. Cellulosomes are large protein networks used by certain bacteria to bind and digest cellulose fibers. The interaction between Xmodule-dockerin B (XMod-DocB) and cohesin E (CohE) is responsible for attaching the cellulosome of human gut bacterium R. champanellensis (Rc.) to the cell wall and therefore is crucial for cellulosome function. SMFS is used to demonstrate that the XMod-DocB:CohE complex can be formed in two different conformations, a behavior known as ‘dual-binding mode’, and dissociates through three pathways with distinct mechanical stabilities under force. The complex preferably populates a high force pathway under increased force loading rate, precisely resembling a catch bond.
In addition to naturally occurring adhesion proteins, the mechanostability of antibodies and alternative scaffolds is also important for their functions in the context of antibody-coated therapeutic nanoparticles. Anticalin is a type of alternative scaffold developed to target various human cell surface receptors and small molecules related to diseases. One of its targets is cytotoxic T-lymphocyte antigen 4 (CTLA-4), an important target for tumor immunotherapy. Using SMFS combined with non-canonical amino acid incorporation and click chemistry, external pulling forces are applied to anticalin from eight different directions to dissociate it from CTLA-4 and characterize the geometric dependency of the unbinding energy landscape. The highest rupture force which is ~100% higher than the least mechanostable pulling geometry is found when pulling from residue 60 of anticalin.
The anisotropic response of proteins to mechanical forces can also be used to engineer naturally occurring protein-ligand systems and change their mechanical properties. Another Doc:Coh system from Rc., DocG:CohE complex, dissociates in two pathways under force. The pulling geometry affects the rupture force in both pathways as well as the rate of entering each pathway. When pulling from residue 13 of CohE, the complex exhibits a catch bond behavior, which is distinct from other measured pulling geometries, including the native pulling geometry.
In summary, SMFS is used here both to understand the underlying mechanisms of mechanostable protein-ligand complexes and to engineer them for higher mechanical stabilities as well as unique behaviors such as catch bonding
Selective Transport of Functional Polymer-Hybrid Vesicles into Cell Nuclei
Get PDFThe cell nucleus is the ultimate target for many therapeutic treatments including cancer, brain disorders and heart dysfunction. Therefore, organelle-specific nanocarriers (NCs) are highly sought after for delivering sufficient concentrations of the active therapeutic agent in situ. This requires the NCs to interact with the nucleocytoplasmic transport (NCT) to enter nuclear pore complexes (NPCs). Yet, little is known as to how NCs infiltrate this vital intracellular barrier to enter the nuclear interior. Furthermore, it is poorly understood how the physico-chemical NC properties influence this process.
Here, ∼50 nm-sized synthetic NCs were developed based on polymer-hybrid vesicles, known as polymersomes. Following a bottom-up approach, biocompatible and amphiphilic PMOXA-PDMS-PMOXA triblock copolymers were self-assembled into NCs and surface-conjugated with nuclear localization sequences (NLS). Those NLS-NCs represent ideal candidates to study NCT, as they remain structurally intact during nuclear import due to the enhanced polymersome membrane stability and strength as compared to liposomes. Moreover, the NLS surface tags authenticate NCs as nucleus specific and enable the NCT mediated import. Applying a so-called film rehydration method permits encapsulating the hydrophilic model drug Ruthenium Red inside the aqueous vesicle cavity and post-treatment with Bodipy 630/650 allows intercalating a lipophilic model drug into the membrane of the same NC. The encapsulated drugs are consequently protected against premature degradation and carried together to the nucleus.
In addition, NLS-NCs were used as large cargoes to study NCT mechanisms. Detailed chemical, biophysical and cellular analysis show that karyopherin receptors (Kaps) are required to bind and escort NLS-NCs through NPCs while Ran guanosine triphosphate (RanGTP) promotes their release from NPCs into the nuclear interior. Ultrastructural analysis by transmission electron microscopy further resolves NLS-NCs on transit in NPCs and inside the nucleus. By elucidating their ability to utilize NCT, these findings demonstrate the efficacy of polymersomes to deliver encapsulated payloads directly into cell nuclei
Multivariate Effects of Brain Micro-environmental Constituents on Amyloid Proteins
Get PDFAberrant accumulation of amyloid proteins into different kinds of aggregates is closely related to a cluster of disorders, called protein misfolding diseases. Various micro-environmental constituents in brains modulate amyloid proteins and have been drawing extensive attention in the field of amyloid study. However, the effects of these micro-environmental constituents in brain such as proteins (chaperone, amyloid, and Apolipoprotein E (ApoE) proteins), lipids, metal ions, pH, and ionic strength, have been only investigated separately, with no consensus achieved. In this thesis work, I studied how different combinations of these micro-environmental constituents affect amyloid proteins. Specifically, the cross-interactions between Apolipoprotein E and various amyloid proteins were reviewed in Chapter 2; the covariant effects of Hsp90/ATP and pH/ionic strength/zinc ions on Aβ40 peptides were investigated in Chapter 3 and Chapter 4, respectively; and how lipid and copper ions modulate alpha-synuclein (α-Syn) protein was studied in Chapter 5. The results are as follows: (1) ApoE interacts with different amyloid proteins and one specific amyloid protein can interact with both ApoE and other amyloid proteins; (2) ATP impedes the inhibitory effect of Hsp90 on Aβ40 fibrillation; (3) pH, salt, and zinc ions possess significantly different effects on Aβ40 fibrillation, depending on the specific combination of these three micro-environmental constituents, and the morphology of Aβ40 aggregates can be modulated by the presence of salt; (4) α-Syn proteins modulate the phase behavior of lipidic cubic phase (LCP) and copper ions at micromolar range counteract the effect of α-Syn on the phase behavior of LCP. These results can, to some extent, explain the discrepancy in this field and draw more attention to the multivariate effects of different micro-environmental constituents in brains on amyloid proteins
