Otitis media (OM) is a common pediatric disease for which systemic antibiotics are often prescribed. While local treatment would avoid the systemic treatment side-effects, the tympanic membrane (TM) represents an impenetrable barrier unless surgically breached. We hypothesized that the TM might harbor innate biological mechanisms that could mediate trans-TM transport. We used two M13-bacteriophage display biopanning strategies to search for mediators of trans-TM transport. First, aliquots of linear phage library displaying 10(10th) 12mer peptides were applied on the TM of rats with active bacterial OM. The middle ear (ME) contents were then harvested, amplified and the preparation re-applied for additional rounds. Second, the same naïve library was sequentially screened for phage exhibiting TM binding, internalization and then transit. Results revealed a novel set of peptides that transit across the TM to the ME in a time and temperature dependent manner. The peptides with highest transport capacities shared sequence similarities. Historically, the TM was viewed as an impermeable barrier. However, our studies reveal that it is possible to translocate peptide-linked small particles across the TM. This is the first comprehensive biopanning for the isolation of TM transiting peptidic ligands. The identified mechanism offers a new drug delivery platform into the ME.

The metabolic enzyme cytidine triphosphate synthase has recently been found to form micrometer-sized filamentous structures termed cytoophidia, which are evolutionarily conserved across prokaryotes and eukaryotes. The cytoophidium represents a novel type of membraneless organelle and behaves dynamically inside the cell. The question of how cytoophidia transport is mediated, however, remains unanswered. For the first time, we detected in this study the active transport of cytoophidia, taking advantage of the fission yeast Schizosaccharomyces pombe as an excellent model for studying membraneless organelles. We demonstrated that actin filaments, not microtubules, are responsible for this transport. Furthermore, we determined that Myo52, a type of myosin V, is required for the active transport of cytoophidia. These results reveal the major players critical to the dynamics of cytoophidia and extend our understanding of intracellular transport of membraneless organelles.-Li, H., Ye, F., Ren, J.-Y., Wang, P.-Y., Du, L.-L., Liu, J.-L. Active transport of cytoophidia in Schizosaccharomyces pombe.

Phosphatidylethanolamine (PE) is an essential phospholipid for mitochondrial functions and is synthesized mainly by phosphatidylserine (PS) decarboxylase at the mitochondrial inner membrane. In Saccharomyces cerevisiae, PS is synthesized in the endoplasmic reticulum (ER), such that mitochondrial PE synthesis requires PS transport from the ER to the mitochondrial inner membrane. Here, we provide evidence that Ups2-Mdm35, a protein complex localized at the mitochondrial intermembrane space, mediates PS transport for PE synthesis in respiration-active mitochondria. UPS2- and MDM35-null mutations greatly attenuated conversion of PS to PE in yeast cells growing logarithmically under nonfermentable conditions, but not fermentable conditions. A recombinant Ups2-Mdm35 fusion protein exhibited phospholipid-transfer activity between liposomes in vitro. Furthermore, UPS2 expression was elevated under nonfermentable conditions and at the diauxic shift, the metabolic transition from glycolysis to oxidative phosphorylation. These results demonstrate that Ups2-Mdm35 functions as a PS transfer protein and enhances mitochondrial PE synthesis in response to the cellular metabolic state.

The role of mechanical forces driving kidney epithelial fluid transport and morphogenesis in kidney diseases is unclear. Here, using a microfluidic platform to recapitulate fluid transport activity of kidney cells, we report that renal epithelial cells can actively generate hydraulic pressure gradients across the epithelium. The fluidic flux declines with increasing hydraulic pressure until a stall pressure, in a manner similar to mechanical fluid pumps. For normal human kidney cells, the fluidic flux is from apical to basal, and the pressure is higher on the basal side. For human Autosomal Dominant Polycystic Kidney Disease cells, the fluidic flux is reversed from basal to apical. Molecular and proteomic studies reveal that renal epithelial cells are sensitive to hydraulic pressure gradients, changing gene expression profiles and spatial arrangements of ion exchangers and the cytoskeleton in different pressure conditions. These results implicate mechanical force and hydraulic pressure as important variables during kidney function and morphological change, and provide insights into pathophysiological mechanisms underlying the development and transduction of hydraulic pressure gradients.

Self-propelling micromotors are emerging as a promising micro- and nanoscale tool for single-cell analysis. We have recently shown that the field gradients necessary to manipulate matter via dielectrophoresis can be induced at the surface of a polarizable active ("self-propelling") metallodielectric Janus particle (JP) under an externally applied electric field, acting essentially as a mobile floating microelectrode. Here, we successfully demonstrated that the application of an external electric field can singularly trap and transport bacteria and can selectively electroporate the trapped bacteria. Selective electroporation, enabled by the local intensification of the electric field induced by the JP, was obtained under both continuous alternating current and pulsed signal conditions. This approach is generic and applicable to bacteria and JP, as well as a wide range of cell types and micromotor designs. Hence, it constitutes an important and novel experimental tool for single-cell analysis and targeted delivery.

Nanoscale transport through nanopores and live-cell membranes plays a vital role in both key biological processes as well as biosensing and DNA sequencing. Active translocation of DNA through these nanopores usually needs enzyme assistance. Here we present a nanopore derived from truncated helicase E1 of bovine papillomavirus (BPV) with a lumen diameter of c.a. 1.3 nm. Cryogenic electron microscopy (cryo-EM) imaging and single channel recording confirm its insertion into planar lipid bilayer (BLM). The helicase nanopore in BLM allows the passive single-stranded DNA (ssDNA) transport and retains the helicase activity in vitro. Furthermore, we incorporate this helicase nanopore into the live cell membrane of HEK293T cells, and monitor the ssDNA delivery into the cell real-time at single molecule level. This type of nanopore is expected to provide an interesting tool to study the biophysics of biomotors in vitro, with potential applications in biosensing, drug delivery and real-time single cell analysis.

Understanding the active transport of substrates by the kidney in the renal proximal convoluted tubule is crucial for drug development and for studying kidney diseases. Currently, cell-based assays are applied for this this purpose, however, differences between assays and the body are common, indicating the importance of in vitro-in vivo discrepancies. Several studies have suggested that 3D cell cultures expose cells to a more physiological environments, thus, providing more accurate cell function results. To mimic the renal proximal tubule, we have developed a custom-made renal module (RM), containing a single polypropylene hollow fibre (Plasmaphan P1LX, 3M) that serves as a porous scaffold and compared to conventional Transwell cell-based bidirectional transport studies. In addition, a constant flow of media, exposed cells to a physiological shear stress of 0.2 dyne/cm2. MDCK-Mdr1a cells, overexpressing the rat Mdr1a (P-gp) transporter, were seeded onto the HF membrane surface coated with the basement membrane matrix Geltrex which facilitated cell adhesion and tight junction formation. Cells were then seeded into the HF lumen where attachment and tight junction formation were evaluated by fluorescence microscopy while epithelial barrier integrity under shear stress was shown to be achieved by day 7. qPCR results have shown significant changes in gene expression compared to cells grown on Transwells. Kidney injury marker such as KIM-1 and the hypoxia marker CA9 have been downregulated, while the CD133 (Prominin-1) microvilli marker has shown a fivefold upregulation. Furthermore, the renal transporter P-gp expression has been downregulated by 50%. Finally, bidirectional assays have shown that cells grown in the RM were able to reabsorb albumin with a higher efficiency compared to Transwell cell cultures while efflux of the P-gp-specific substrates Hoechst and Rhodamine 123 was decreased. These results further support the effect of the microenvironment and fluidic shear stress on cell function and gene expression. This can serve as the basis for the development of a microphysiological renal model for drug transport studies.

Heterogeneous distribution of components in the biological membrane is critical in the process of cell polarization. However, little is known about the mechanisms that can generate and maintain the heterogeneous distribution of the membrane components. Here, we report that the propagating wave patterns of the bacterial Min proteins can impose steric pressure on the membrane, resulting in transport and directional accumulation of the component in the membrane. Therefore, the membrane component waves represent transport of the component in the membrane that is caused by the steric pressure gradient induced by the differential levels of binding and dissociation of the Min proteins in the propagating waves on the membrane surface. The diffusivity, majorly influenced by the membrane anchor of the component, and the repulsed ability, majorly influenced by the steric property of the membrane component, determine the differential spatial distribution of the membrane component. Thus, transportation of the membrane component by the Min proteins follows a simple physical principle, which resembles a linear peristaltic pumping process, to selectively segregate and maintain heterogeneous distribution of materials in the membrane. VIDEO ABSTRACT.

The passive diffusion performance of nanocarriers results in inefficient drug transport across multiple biological barriers and consequently cancer therapy failure. Here, a magnetically driven amoeba-like nanorobot (amNR) is presented for whole-process active drug transport. The amNR is actively extravasated from blood vessels and penetrated into deep tumor tissue through a magnetically driven deformation effect. Moreover, the acidic microenvironment of deep tumor tissue uncovers the masked targeting ligand of amNR to achieve active tumor cell uptake. Furthermore, the amNR rapidly releases the encapsulated doxorubicin (DOX) after alternating magnetic field application. The amNRs eventually deliver DOX into ≈92.3% of tumor cells and completely delay tumor growth with an inhibition rate of 96.1%. The deformable amNRs, with the assistance of magnetic field application, provide a facile strategy for whole-process active drug transport.

Utilization of active colloids to transport both biological and inorganic cargo has been widely examined in the context of applications ranging from targeted drug delivery to sample analysis. In general, carriers are customized to load one specific target via a mechanism distinct from that driving the transport. Here we unify these tasks and extend loading capabilities to include on-demand selection of multiple nano/micro-sized targets without the need for pre-labelling or surface functionalization. An externally applied electric field is singularly used to drive the active cargo carrier and transform it into a mobile floating electrode that can attract (trap) or repel specific targets from its surface by dielectrophoresis, enabling dynamic control of target selection, loading and rate of transport via the electric field parameters. In the future, dynamic selectivity could be combined with directed motion to develop building blocks for bottom-up fabrication in applications such as additive manufacturing and soft robotics.

Myosins are actin-based molecular motors which are enzymatically adapted for their cellular functions such as transportation and membrane tethering. Human Myo19 affects mitochondrial motility, and promotes their localization to stress-induced filopodia. Therefore, studying Myo19 enzymology is essential to understand how this motor may facilitate mitochondrial motility. Towards this goal, we have purified Myo19 motor domain (Myo19-3IQ) from a human-cell expression system and utilized transient kinetics to study the Myo19-3IQ ATPase cycle. We found that Myo19-3IQ exhibits noticeable conformational changes (isomerization steps) preceding both ATP and ADP binding, which may contribute to nucleotide binding regulation. Notably, the ADP isomerization step and subsequent ADP release contribute significantly to the rate-limiting step of the Myo19-3IQ ATPase cycle. Both the slow ADP isomerization and ADP release prolong the time Myo19-3IQ spend in the strong actin binding state and hence contribute to its relatively high duty ratio. However, the predicted duty ratio is lower than required to support motility as a monomer. Therefore, it may be that several Myo19 motors are required to propel mitochondria movement on actin filaments efficiently. Finally, we provide a model explaining how Myo19 translocation may be regulated by the local ATP/ADP ratio, coupled to the mitochondria presence in the filopodia.

Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo-EM and by imaging toxin import, we uncover the mechanism by which the Tol-dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9's disordered N-terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton-motive force, which is delivered by the TolQ-TolR-TolA-TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol- and Ton-dependent bacteriocins cross the bacterial outer membrane.

Through billions of years of evolution, microorganisms mastered unique swimming behaviors to thrive in complex fluid environments. Limitations in nanofabrication have thus far hindered the ability to design and program synthetic swimmers with the same abilities. Here we encode multi-behavioral responses in microscopic self-propelled tori using nanoscale 3D printing. We show experimentally and theoretically that the tori continuously transition between two primary swimming modes in response to a magnetic field. The tori also manipulated and transported other artificial swimmers, bimetallic nanorods, as well as passive colloidal particles. In the first behavioral mode, the tori accumulated and transported nanorods; in the second mode, nanorods aligned along the tori's self-generated streamlines. Our results indicate that such shape-programmed microswimmers have a potential to manipulate biological active matter, e.g. bacteria or cells.

Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes.

Nature encapsulates reactions within membrane-bound compartments, affording sequential and spatial control over biochemical reactions. Droplet Interface Bilayers are evolving into a valuable platform to mimic this key biological feature in artificial systems. A major issue is manipulating flow across synthetic bilayers. Droplet Interface Bilayers must be functionalised, with seminal work using membrane-inserting toxins, ion channels and pumps illustrating the potential. Specific transport of biomolecules, and notably transport against a concentration gradient, across these bilayers has yet to be demonstrated. Here, we successfully incorporate the archetypal Major Facilitator Superfamily transporter, lactose permease, into Droplet Interface Bilayers and demonstrate both passive and active, uphill transport. This paves the way for controllable transport of sugars, metabolites and other essential biomolecular substrates of this ubiquitous transporter superfamily in DIB networks. Furthermore, cell-free synthesis of lactose permease during DIB formation also results in active transport across the interface bilayer. This adds a specific disaccharide transporter to the small list of integral membrane proteins that can be synthesised via in vitro transcription/translation for applications of DIB-based artificial cell systems. The introduction of a means to promote specific transport of molecules across Droplet Interface Bilayers against a concentration gradient gives a new facet to droplet networks.

P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate.

The syntheses and preliminary biological evaluation of conjugates of a synthetic isocyanurate-based trihydroxamate siderophore with two antifungal agents, 5-FU (conjugate 9) and norneoenactin (conjugate 12), and a macrolide antibiotic, erythromycylamine (conjugate 18), are described. A 19F NMR study was used to determine the hydrolytic stability of conjugate 9 under assay conditions. Preliminary biological studies with ferric complexes of conjugates 9 and 12 indicated that these antifungal agents are recognized by Candida and perhaps are actively transported into the cell by the siderophore-transport mechanisms. While conjugate 18 did not show any significant antibacterial activity, presumably due to size restriction, the 5-FU conjugate 9 appeared to be moderately active against a variety of Gram-positive strains, and was more active than the 5-FC control against some strains of Staphylococcus.

Loss of the AE3 Cl-/HCO3- exchanger (Slc4a3) in mice causes an impaired cardiac force-frequency response and heart failure under some conditions but the mechanisms are not known. To better understand the functions of AE3, we performed RNA Seq analysis of AE3-null and wild-type mouse hearts and evaluated the data with respect to three hypotheses (CO2 disposal, facilitation of Na+-loading, and recovery from an alkaline load) that have been proposed for its physiological functions. Gene Ontology and PubMatrix analyses of differentially expressed genes revealed a hypoxia response and changes in vasodilation and angiogenesis genes that strongly support the CO2 disposal hypothesis. Differential expression of energy metabolism genes, which indicated increased glucose utilization and decreased fatty acid utilization, were consistent with adaptive responses to perturbations of O2/CO2 balance in AE3-null myocytes. Given that the myocardium is an obligate aerobic tissue and consumes large amounts of O2, the data suggest that loss of AE3, which has the potential to extrude CO2 in the form of HCO3-, impairs O2/CO2 balance in cardiac myocytes. These results support a model in which the AE3 Cl-/HCO3- exchanger, coupled with parallel Cl- and H+-extrusion mechanisms and extracellular carbonic anhydrase, is responsible for active transport-mediated disposal of CO2.

Transmitter exocytosis from the neuronal soma is evoked by brief trains of high frequency electrical activity and continues for several minutes. Here we studied how active vesicle transport towards the plasma membrane contributes to this slow phenomenon in serotonergic leech Retzius neurons, by combining electron microscopy, the kinetics of exocytosis obtained from FM1-43 dye fluorescence as vesicles fuse with the plasma membrane, and a diffusion equation incorporating the forces of local confinement and molecular motors. Electron micrographs of neurons at rest or after stimulation with 1 Hz trains showed cytoplasmic clusters of dense core vesicles at 1.5±0.2 and 3.7±0.3 µm distances from the plasma membrane, to which they were bound through microtubule bundles. By contrast, after 20 Hz stimulation vesicle clusters were apposed to the plasma membrane, suggesting that transport was induced by electrical stimulation. Consistently, 20 Hz stimulation of cultured neurons induced spotted FM1-43 fluorescence increases with one or two slow sigmoidal kinetics, suggesting exocytosis from an equal number of vesicle clusters. These fluorescence increases were prevented by colchicine, which suggested microtubule-dependent vesicle transport. Model fitting to the fluorescence kinetics predicted that 52-951 vesicles/cluster were transported along 0.60-6.18 µm distances at average 11-95 nms(-1) velocities. The ATP cost per vesicle fused (0.4-72.0), calculated from the ratio of the ΔG(process)/ΔG(ATP), depended on the ratio of the traveling velocity and the number of vesicles in the cluster. Interestingly, the distance-dependence of the ATP cost per vesicle was bistable, with low energy values at 1.4 and 3.3 µm, similar to the average resting distances of the vesicle clusters, and a high energy barrier at 1.6-2.0 µm. Our study confirms that active vesicle transport is an intermediate step for somatic serotonin exocytosis by Retzius neurons and provides a quantitative method for analyzing similar phenomena in other cell types.

Antibody-drug conjugates (ADCs) represent a new class of cancer therapeutics that enable targeted delivery of cytotoxic drugs to cancer cells. Although clinical efficacy has been demonstrated for ADC therapies, resistance to these conjugates may occur. Recently, SLC46A3, a lysosomal membrane protein, was revealed to regulate the efficacy of trastuzumab emtansine (T-DM1), a noncleavable ADC that has been widely used for treating breast cancer. However, the role of SLC46A3 in mediating T-DM1 cytotoxicity remains unclear. In this study, we discovered the function of SLC46A3 as a novel proton-coupled steroid conjugate and bile acid transporter. SLC46A3 preferentially recognized lipophilic steroid conjugates and bile acids as endogenous substrates. In addition, we found that SLC46A3 directly transports Lys-SMCC-DM1, a major catabolite of T-DM1, and potent SLC46A3 inhibitors attenuate the cytotoxic effects of T-DM1, suggesting a role in the escape of Lys-SMCC-DM1 from the lysosome into the cytoplasm. Our findings reveal the molecular mechanism by which T-DM1 kills cancer cells and may contribute to the rational development of ADCs that target SLC46A3.