Fluorescence Interference Contrast Microscopy Research Articles

Drunken lipid membranes, not drunken SNARE proteins, promote fusion in a model of neurotransmitter release.

Alcohol affects many neuronal proteins that are upstream or down-stream of synaptic vesicle fusion and neurotransmitter release. Less well studied is alcohol’s effect on the fusion machinery including SNARE proteins and lipid membranes. Using a SNARE-driven fusion assay we show that fusion probability is significantly increased at 0.4% v/v (68 mM) ethanol; but not with methanol up to 10%. Ethanol appears to act directly on membrane lipids since experiments focused on protein properties [circular dichroism spectrometry, site-directed fluorescence interference contrast (sdFLIC) microscopy, and vesicle docking results] showed no significant changes up to 5% ethanol, but a protein-free fusion assay also showed increased lipid membrane fusion rates with 0.4% ethanol. These data show that the effects of high physiological doses of ethanol on SNARE-driven fusion are mediated through ethanol’s interaction with the lipid bilayer of membranes and not SNARE proteins, and that methanol affects lipid membranes and SNARE proteins only at high doses.

Visualizing and Quantifying Wettability Alteration by Silica Nanofluids.

An aqueous suspension of silica nanoparticles or nanofluid can alter the wettability of surfaces, specifically by making them hydrophilic and oil-repellent under water. Wettability alteration by nanofluids has important technological applications, including for enhanced oil recovery and heat transfer processes. A common way to characterize the wettability alteration is by measuring the contact angles of an oil droplet with and without nanoparticles. While easy to perform, contact angle measurements do not fully capture the wettability changes to the surface. Here, we employed several complementary techniques, such as cryo-scanning electron microscopy, confocal fluorescence and reflection interference contrast microscopy, and droplet probe atomic force microscopy (AFM), to visualize and quantify the wettability alterations by fumed silica nanoparticles. We found that nanoparticles adsorbed onto glass surfaces to form a porous layer with hierarchical micro- and nanostructures. The porous layer can trap a thin water film, which reduces contact between the oil droplet and the solid substrate. As a result, even a small addition of nanoparticles (0.1 wt %) lowers the adhesion force for a 20 μm sized oil droplet by more than 400 times from 210 ± 10 to 0.5 ± 0.3 nN as measured by using droplet probe AFM. Finally, we show that silica nanofluids can improve oil recovery rates by 8% in a micromodel with glass channels that resemble a physical rock network.

Platelet adhesion and aggregate formation controlled by immobilised and soluble VWF

BackgroundIt has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF’s mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis.ResultsCombining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM), here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively ‘masks’ the potential between the platelet glycoprotein (GP) Ib and the VWF A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics.ConclusionsThese data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.

Prenylated phenolics as promising candidates for combination antibacterial therapy: Morusin and kuwanon G

Combination of antibiotics with natural products is a promising strategy for potentiating antibiotic activity and overcoming antibiotic resistance. The purpose of the present study was to investigate whether morusin and kuwanon G, prenylated phenolics in Morus species, have the ability to enhance antibiotic activity and reverse antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis. Commonly used antibiotics (oxacillin, erythromycin, gentamicin, ciprofloxacin, tetracycline, clindamycin) were selected for the combination studies. Checkerboard and time-kill assays were used to investigate potential bacteriostatic and bactericidal synergistic interactions, respectively between morusin or kuwanon G and antibiotics. According to both fractional inhibitory concentration index and response surface models, twenty combinations (14 morusin-antibiotic combinations, six kuwanon G-antibiotic combinations) displaying bacteriostatic synergy were identified, with 4–512-fold reduction in the minimum inhibitory concentration values of antibiotics in combination. Both morusin and kuwanon G reversed oxacillin resistance of methicillin-resistant Staphylococcus aureus. In addition, morusin reversed tetracycline resistance of Staphylococcus epidermidis. At half of the minimum inhibitory concentrations, combinations of morusin with oxacillin or gentamicin showed bactericidal synergy against methicillin-resistant Staphylococcus aureus. Fluorescence and differential interference contrast microscopy and scanning electron microscopy showed an increase in the membrane permeability and massive leakage of cellular content in methicillin-resistant Staphylococcus aureus exposed to morusin or kuwanon G. Overall, our findings strongly indicate that both prenylated compounds are good candidates for the development of novel antibacterial combination therapies.

A high-throughput microfluidic method for fabricating aligned collagen fibrils to study Keratocyte behavior.

In vivo, keratocytes are surrounded by aligned type I collagen fibrils that are organized into lamellae. A growing body of literature suggests that the unique topography of the corneal stroma is an important regulator of keratocyte behavior. In this study we describe a microfluidic method to deposit aligned fibrils of type I collagen onto glass coverslips. This high-throughput method allowed for the simultaneous coating of up to eight substrates with aligned collagen fibrils. When these substrates were integrated into a PDMS microwell culture system they provided a platform for high-resolution imaging of keratocyte behavior. Through the use of wide-field fluorescence and differential interference contrast microscopy, we observed that the density of collagen fibrils deposited was dependent upon both the perfusion shear rate of collagen and the time of perfusion. In contrast, a similar degree of fibril alignment was observed over a range of shear rates. When primary normal rabbit keratocytes (NRK) were seeded on substrates with a high density of aligned collagen fibrils and cultured in the presence of platelet derived growth factor (PDGF) the keratocytes displayed an elongated cell body that was co-aligned with the underlying collagen fibrils. In contrast, when NRK were cultured on substrates with a low density of aligned collagen fibrils, the cells showed no preferential orientation. These results suggest that this simple and inexpensive method can provide a general platform to study how simultaneous exposure to topographical and soluble cues influence cell behavior.

A Brownian Ratchet Model Explains the Biased Sidestepping of Single-Headed Kinesin-3 KIF1A

The kinesin-3 motor KIF1A is involved in long-ranged axonal transport in neurons. To ensure vesicular delivery, motors need to navigate the microtubule lattice and overcome possible roadblocks along the way. The single-headed form of KIF1A is a highly diffusive motor that has been shown to be a prototype of a Brownian motor by virtue of a weakly bound diffusive state to the microtubule. Recently, groups of single-headed KIF1A motors were found to be able to sidestep along the microtubule lattice, creating left-handed helical membrane tubes when pulling on giant unilamellar vesicles in vitro. A possible hypothesis is that the diffusive state enables the motor to explore the microtubule lattice and switch protofilaments, leading to a left-handed helical motion. Here, we study the longitudinal rotation of microtubules driven by single-headed KIF1A motors using fluorescence-interference contrast microscopy. We find an average rotational pitch of ≃1.5μm, which is remarkably robust to changes in the gliding velocity, ATP concentration, microtubule length, and motor density. Our experimental results are compared to stochastic simulations of Brownian motors moving on a two-dimensional continuum ratchet potential, which quantitatively agree with the fluorescence-interference contrast experiments. We find that single-headed KIF1A sidestepping can be explained as a consequence of the intrinsic handedness and polarity of the microtubule lattice in combination with the diffusive mechanochemical cycle of the motor.

Interplay between Spinodal Decomposition and Gelation and Their Role in Two- and Three-Dimensional Pattern Formation at the Gelatin Gel Surface

Understanding the mechanisms underlying the spontaneous formation of Liesegang patterns based on the prenucleation and postnucleation models is of critical importance for understanding pattern formation in nature. In contrast to the rapid experimental and theoretical advances in understanding within the framework of the prenucleation model, discussion of the postnucleation model is mostly limited to numerical analysis. To construct a standard model for a chemical experiment discussed in terms of the postnucleation model, we have investigated the pattern formation mechanism in a mixed system containing gelatin, starch, and sugar. Fluorescence and differential interference contrast microscopy revealed a process of two-dimensional spinodal decomposition into gelatin-rich and gelatin-poor phases. Since the gelation temperature of the gelatin-rich phase was higher, spatially periodic gelation proceeded selectively in the gelatin-rich phase. As both two-dimensional spinodal decomposition and three-dimensional g...

Kinesin motor density and dynamics in gliding microtubule motility

Kinesin motors and their associated filaments, microtubules, are essential to many biological processes. The motor and filament system can be reconstituted in vitro with the surface-adhered motors transporting the filaments along the surface. In this format, the system has been used to study active self-assembly and to power microdevices or perform analyte detection. However, fundamental properties of the system, such as the spacing of the kinesin motors bound to the microtubule and the dynamics of binding, remain poorly understood. We show that Fluorescence Interference Contrast (FLIC) microscopy can illuminate the exact height of the microtubule, which for a sufficiently low surface density of kinesin, reveals the locations of the bound motors. We examine the spacing of the kinesin motors on the microtubules at various kinesin surface densities and compare the results with theory. FLIC reveals that the system is highly dynamic, with kinesin binding and unbinding along the length of the microtubule as it is transported along the surface.

Combining Electrical and Optical Measurements to Reveal the Structure-Function Relationship of Voltage-Gated Potassium Channels

Although the structure of the voltage-gated potassium channel KvAP has been resolved more than a decade ago, there is still controversy about their precise gating mechanism. The discrepancies among proposed models result in part from differences in experimental techniques: electrical measurements of channels in lipid membranes to study their function versus determination of the structure of channels purified using detergents. To be able to combine functional and structural measurements, we developed a second-generation membrane interferometer in which both electrophysiology measurements and high-resolution fluorescence microscopy imaging can be performed on ion channels reconstituted in a lipid membrane, a much advanced design over the original (Ganesan et al., Proc. Natl. Acad. Sci. 106, 2008). In the membrane interferometer, a freestanding bilayer is formed over a micropore that is positioned above a reflective mirror. The mirror allows the use of Fluorescence Interference Contrast (FLIC) and Variable Incidence Angle-FLIC (VIA-FLIC) microscopy, two surface characterization techniques that precisely locate the height of fluorescent objects relative to the mirror with nanometer resolution. KvAP overexpressed in E. coli is detergent-free extracted using polymer nanodiscs. We show that functional KvAP can be reconstituted in black lipid membranes and freestanding lipid bilayers on the membrane interferometer. No differences in gating properties were found between KvAP extracted by polymer nanodiscs versus detergents. We label the channels with a fluorescent tag at different positions at the S3 or S4 strand. Our goal is to correlate the change in position of the S3 and S4 strands measured using FLIC microscopy with single-channel electrical measurements to reveal the structure-function relationship of KvAP. Progress towards this goal will be described.

Working stroke of the kinesin-14, ncd, comprises two substeps of different direction

Single-molecule experiments have been used with great success to explore the mechanochemical cycles of processive motor proteins such as kinesin-1, but it has proven difficult to apply these approaches to nonprocessive motors. Therefore, the mechanochemical cycle of kinesin-14 (ncd) is still under debate. Here, we use the readout from the collective activity of multiple motors to derive information about the mechanochemical cycle of individual ncd motors. In gliding motility assays we performed 3D imaging based on fluorescence interference contrast microscopy combined with nanometer tracking to simultaneously study the translation and rotation of microtubules. Microtubules gliding on ncd-coated surfaces rotated around their longitudinal axes in an [ATP]- and [ADP]-dependent manner. Combined with a simple mechanical model, these observations suggest that the working stroke of ncd consists of an initial small movement of its stalk in a lateral direction when ADP is released and a second, main component of the working stroke, in a longitudinal direction upon ATP binding.

Fluorescence Interference Contrast Microscopy (FLIC) - A New Tool to Study the Collective Motor Dynamics

Further development of an accurate model of actomyosin network function requires detailed knowledge of the interaction between actin and myosin. The majority of data demonstrating the function of myosins has been obtained either in a single molecule fashion (where the isolated motors have been studied) or in a gliding filament assay (where multiple motors work collectively). Here, I present a technique that joins collective motor mechanics with single molecule detection of binding events, using Fluorescence Interference Contrast Microscopy (FLIC).In the FLIC assay, the distance between a fluorescently labeled actin filament and a reflective surface of a slide can be determined. The locations of myosin molecules that tether the actin filament to the surface are clearly visible. An attachment lifetime of each actin-motor interaction can be easily extracted from these data. The length of actin between two myosins (buckled or under tension) provides information about the relative speed of the two myosins. Moreover, segments of actin under tension and compression transduce different amounts of force to the myosins at each end.Our data show that the attachment lifetimes of Myosin 6 and Non-Muscle Myosin IIB are much longer in gliding filament assay than the attachment lifetimes previously obtained in single molecule bead assay. We argue that this difference, at least partially, can be attributed to mechanical coupling through the actin filament.

FLIC Microscopy Reveals Different Conformational States of Syntaxin 1a in Supported Lipid Bilayers

The fusion of synaptic vesicles with the presynaptic membrane is a fast and highly regulated process that is catalyzed by the exocytotic SNAREs synaptobrevin-2 (VAMP-2), SNAP-25, and syntaxin-1a (Syx1a). In addition to the SNAREs a number of protein players that regulate the fusion process have been identified and characterized. Despite the tremendous progress in this field we are still missing a molecular timeline that leads from docking of synaptic vesicles to the plasma membrane, to priming of the fusion machinery and eventually to fusion of the two membranes once an action potential reaches the synaptic terminal. One of the difficulties in approaching these questions arises from the fact that the lipid environment plays an active role in regulating this protein driven process.Reconstituting SNAREs into supported lipid bilayers allows us to characterize the state of these proteins in different lipid environments. Using fluorescence interference contrast (FLIC) microscopy we measure distances of specific residues in Syx1a from the membrane surface. Here, we report how regulatory proteins like Munc18 and Munc13 modulate the state of Syx1a in the presence and absence of SNAP-25.

Combining Fluorescence Microscopy on Freestanding Lipid Bilayers with Electrical Measurements

The structure, precise operation mechanism and dynamics of ion channels have not yet been elucidated because linking the structure and structure changes to function has remained challenging. We are developing new methods that allow the structure of membrane proteins to be studied in a controlled, native-like environment by simultaneously probing their structure and function. We report a second-generation membrane interferometer (Ganesan et al., Proc. Natl. Acad. Sci. 106, 2008) in which electrophysiology measurements can be performed simultaneously with high-resolution fluorescence microscopy imaging. In the membrane interferometer, a freestanding bilayer is formed over a micropore that is positioned approximately 400 nanometer above a reflective mirror. The mirror allows the use of Fluorescence Interference Contrast microscopy (FLIC) and Variable Incidence Angle-FLIC (VIA-FLIC), two surface characterization techniques that precisely locate the height of fluorescent objects relative to the silicon surface with nm resolution. The second-generation design provides electrical access on both sides and is meant to create a more planar bilayer than the earlier design. Electrophysiology measurements revealed formation of freestanding bilayers with giga-Ohm seals over the micropores that are stable for over 24 hrs. As an initial test we incorporated alpha-hemolysin channels into the bilayers and measured their activity using electrophysiology, while simultaneously imaging the lipid bilayer by FLIC to analyze its curvature. Progress on the incorporation of other fluorescently labeled ion channels will be described.

Three-Dimensional Motility of the Highly Processive Kinesin-8 Along the Microtubule Lattice

During mitosis, kinesin-8 motors play a key role in regulating the spindle length based to their depolymerization activity at microtubule plus-ends. In order to reach the plus-ends of microtubules, kinesin-8 motors show superprocessive motility. However, the mechanism confering such high motor processivity even on crowded microtubules in the cytoplasm is not well understood. To gain insight into the stepping mechanism of kinesin-8, we explored the three-dimensional motility of yeast kinesin-8, Kip3, along the microtubule lattice in vitro. First, we performed microtubule gliding motility assays on surface coated with Kip3 motors and measured the rotations of gliding microtubules around their longitudinal axis using rhodamine speckled microtubules in combination with fluorescence-interference contrast microscopy [1]. We observed rotations with periodicities significantly smaller than the microtubule supertwist, indicating that the motors do not follow individual protofilaments but rather switch protofilaments stochastically in one direction [2]. Finally, we confirmed this hypothesis by 3D single-molecule stepping assays along freely suspended microtubules by 2D tracking of QDot-conjugated Kip3 motors in combination with parallax [3], a dual-focus imaging technique that provides nanometer information in z-direction.1. Mitra A, Ruhnow F, Nitzsche B, Diez S. Impact-Free Measurement of Microtubule Rotations on Kinesin and Cytoplasmic-Dynein Coated Surfaces. PLoS One. 2015.2. Bormuth V, Nitzsche B, Ruhnow F, Mitra A, Storch M, Rammner B, Howard J, Diez S. The highly processive kinesin-8, Kip3, switches microtubule protofilaments with a bias toward the left. Biophys J. 2012.3. Sun Y, McKenna JD, Murray JM, Ostap EM, Goldman YE. Parallax: high accuracy three-dimensional single molecule tracking using split images. Nano Lett. 2009.

Impact-Free Measurement of Microtubule Rotations on Kinesin and Cytoplasmic-Dynein Coated Surfaces

Knowledge about the three-dimensional stepping of motor proteins on the surface of microtubules (MTs) as well as the torsional components in their power strokes can be inferred from longitudinal MT rotations in gliding motility assays. In previous studies, optical detection of these rotations relied on the tracking of rather large optical probes present on the outer MT surface. However, these probes may act as obstacles for motor stepping and may prevent the unhindered rotation of the gliding MTs. To overcome these limitations, we devised a novel, impact-free method to detect MT rotations based on fluorescent speckles within the MT structure in combination with fluorescence-interference contrast microscopy. We (i) confirmed the rotational pitches of MTs gliding on surfaces coated by kinesin-1 and kinesin-8 motors, (ii) demonstrated the superiority of our method over previous approaches on kinesin-8 coated surfaces at low ATP concentration, and (iii) identified MT rotations driven by mammalian cytoplasmic dynein, indicating that during collective motion cytoplasmic dynein side-steps with a bias in one direction. Our novel method is easy to implement on any state-of-the-art fluorescence microscope and allows for high-throughput experiments.

Enhanced Human Epidermal Growth Factor Receptor 2 Degradation in Breast Cancer Cells by Lysosome-Targeting Gold Nanoconstructs.

This paper describes how gold nanoparticle nanoconstructs can enhance anticancer effects of lysosomal targeting aptamers in breast cancer cells. Nanoconstructs consisting of anti-HER2 aptamer (human epidermal growth factor receptor 2, HApt) densely grafted on gold nanostars (AuNS) first targeted HER2 and then were internalized via HER2-mediated endocytosis. As incubation time increased, the nanoconstruct complexes were found in vesicular structures, starting from early endosomes to lysosomes as visualized by confocal fluorescence and differential interference contrast microscopy. Within the target organelle, lysosomes, HER2 was degraded by enzymes at low pH, which resulted in apoptosis. At specific time points related to the doubling time of the cancer cells, we found that accumulation of HER2-HApt-AuNS complexes in lysosomes, lysosomal activity, and lysosomal degradation of HER2 were positively correlated. Increased HER2 degradation by HApt-AuNS triggered cell death and cell cycle arrest in the G0/G1 phase inhibition of cell proliferation. This work shows how a perceived disadvantage of nanoparticle-based therapeutics-the inability of nanoconstructs to escape from vesicles and thus induce a biological response-can be overcome by both targeting lysosomes and exploiting lysosomal degradation of the biomarkers.

Lipid Membrane Deformation Accompanied by Disk-to-Ring Shape Transition of Cholesterol-Rich Domains.

During vesicle budding or endocytosis, biomembranes undergo a series of lipid- and protein-mediated deformations involving cholesterol-enriched lipid rafts. If lipid rafts of high bending rigidities become confined to the incipient curved membrane topology such as a bud-neck interface, they can be expected to reform as ring-shaped rafts. Here, we report on the observation of a disk-to-ring shape morpho-chemical transition of a model membrane in the absence of geometric constraints. The raft shape transition is triggered by lateral compositional heterogeneity and is accompanied by membrane deformation in the vertical direction, which is detected by height-sensitive fluorescence interference contrast microscopy. Our results suggest that a flat membrane can become curved simply by dynamic changes in local chemical composition and shape transformation of cholesterol-rich domains.

Chasing the Functional Asymmetry between C2A and C2B in Full-Length Synaptotagmin 1 during Ca2+-Dependent Membrane Binding

Synaptotagmin 1 (Syt1) acts as the major calcium sensor in neuronal exocytosis. Syt1 is a synaptic vesicle-anchored membrane protein that contains two tandem Ca2+ binding C2 domains, named C2A and C2B. There is evidence that Syt1 interacts with membrane lipids and SNARE proteins simultaneously to facilitate two key steps during Ca2+-triggered membrane fusion: vesicle binding and content release. Although the overall function of Syt1 has been extensively investigated, the detailed molecular behavior of the single C2A and C2B domains in the neural regulatory process remains unclear. In particular, the differential function of the two C2 domains and how they cooperate in membrane fusion has not been determined.Here we employed EPR spectroscopy, fluorescence interference contrast (FLIC) microscopy and total internal reflection fluorescence (TIRF) microscopy to dissect the state of C2A and C2B domains in full-length Syt1 during Ca2+-dependent membrane binding. CW-EPR lineshapes and power saturation of spin-labeled positions in calcium binding loops of C2A in full-length Syt1 (1-421) and truncated Syt1 (1-266) without C2B domain suggest that C2A domain alone in Syt1 has a stronger membrane binding ability. A TIRF liposome capture assay further reveals that truncated Syt1 has a significant higher initial rate and extent in trans-binding with liposomes than full-length Syt1. Our data indicate that the C2B domain might be involved in some cis-binding thereby reducing total liposome binding. FLIC microscopy validates the strong involvement of C2A during Ca2+-dependent liposome binding.Our detailed structural and functional information thus provides a clue to differential regulatory mechanisms employed by C2A and C2B domains in full-length Syt1 interacting with Ca2+ and key membrane lipids, such as PS and PIP2.

Observing the Mushroom-to-Brush Transition for Kinesin Proteins

The height of polymers grafted to a surface is predicted to be constant at low densities ("mushroom" regime) and increase with the third root of the polymer surface density at high densities ("brush" regime). This mushroom-to-brush transition is explored with kinesin-1 proteins adhered to a surface at controlled densities. The kinesin height is measured by attaching fluorescently labeled microtubules to the kinesins and determining their elevation using fluorescence interference contrast microscopy. Our measurements are consistent with a mushroom regime and a brush regime and a transition near the theoretically predicted density. The mushroom-to-brush transition may play a role in protein behavior in crowded cellular environments and may be exploited as a signal in intracellular regulation and mechanotransduction.

N-Linked glycan site occupancy impacts the distribution of a potassium channel in the cell body and outgrowths of neuronal-derived cells

BackgroundVacancy of occupied N-glycosylation sites of glycoproteins is quite disruptive to a multicellular organism, as underlined by congenital disorders of glycosylation. Since a neuronal component is typically associated with this disease, we evaluated the impact of N-glycosylation processing of a neuronal voltage gated potassium channel, Kv3.1b, expressed in a neuronal-derived cell line, B35 neuroblastoma cells. MethodsTotal internal reflection fluorescence and differential interference contrast microscopy measurements of live B35 cells expressing wild type and glycosylation mutant Kv3.1b proteins were used to evaluate the distribution of the various forms of the Kv3.1b protein in the cell body and outgrowths. Cell adhesion assays were also employed. ResultsMicroscopy images revealed that occupancy of both N-glycosylation sites of Kv3.1b had relatively similar amounts of Kv3.1b in the outgrowth and cell body while vacancy of one or both sites led to increased accumulation of Kv3.1b in the cell body. Further both the fully glycosylated and partially glycosylated N229Q Kv3.1b proteins formed higher density particles in outgrowths compared to cell body. Cellular assays demonstrated that the distinct spatial arrangements altered cell adhesion properties. ConclusionsOur findings provide direct evidence that occupancy of the N-glycosylation sites of Kv3.1b contributes significantly to its lateral heterogeneity in membranes of neuronal-derived cells, and in turn alters cellular properties. General significanceOur study demonstrates that N-glycans of Kv3.1b contain information regarding the association, clustering, and distribution of Kv3.1b in the cell membrane, and furthermore that decreased occupancy caused by congenital disorders of glycosylation may alter the biological activity of Kv3.1b.