3D Single Molecule Tracking with Multifocal Plane Microscopy Reveals Rapid Intercellular Transferrin Transport at Epithelial Cell Barriers

The study of intracellular transport pathways at epithelial cell barriers that line diverse tissue sites is fundamental to understanding tissue homeostasis. A major impediment to investigating such processes at the subcellular level has been the lack of imaging approaches that support fast three-dimensional (3D) tracking of cellular dynamics in thick cellular specimens. Here, we report significant advances in multifocal plane microscopy and demonstrate 3D single molecule tracking of rapid protein dynamics in a 10 micron thick live epithelial cell monolayer. We have investigated the transferrin receptor (TfR) pathway, which is not only essential for iron delivery but is also of importance for targeted drug delivery across cellular barriers at specific body sites, such as the brain that is impermeable to blood-borne substances. Using multifocal plane microscopy, we have discovered a cellular process of intercellular transfer involving rapid exchange of Tf molecules between two adjacent cells in the monolayer. Furthermore, 3D tracking of Tf molecules at the lateral plasma membrane has led to the identification of different modes of endocytosis and exocytosis, which exhibit distinct temporal and intracellular spatial trajectories. These results reveal the complexity of the 3D trafficking pathways in epithelial cell barriers. The methods and approaches reported here can enable the study of fast 3D cellular dynamics in other cell systems and models, and underscore the importance of developing advanced imaging technologies to study such processes.     

Imaging Barriers to Diffusion by Pair Correlation Functions

Molecular diffusion and transport are fundamental processes in physical, chemical, biochemical, and biological systems. However, current approaches to measure molecular transport in cells and tissues based on perturbation methods such as fluorescence recovery after photobleaching are invasive, fluctuation correlation methods are local, and single-particle tracking requires the observation of isolated particles for relatively long periods of time. We propose to detect molecular transport by measuring the time cross-correlation of fluctuations at a pair of locations in the sample. When the points are farther apart than two times the size of the point spread function, the maximum of the correlation is proportional to the average time a molecule takes to move from a specific location to another. We demonstrate the method by simulations, using beads in solution, and by measuring the diffusion of molecules in cellular membranes. The spatial pair cross-correlation method detects barriers to diffusion and heterogeneity of diffusion because the time of the correlation maximum is delayed in the presence of diffusion barriers. This noninvasive, sensitive technique follows the same molecule over a large area, thereby producing a map of molecular flow. It does not require isolated molecules, and thus many molecules can be labeled at the same time and within the point spread function.     

Diffusion barriers in ram and boar sperm plasma membranes: directionality of lipid diffusion across the posterior ring

The plasma membrane of mammalian spermatozoa, like that of other differentiated cells, is compartmentalized into discrete regions or domains that are biochemically and functionally distinct from one another. Physical structures within the membrane, such as the posterior ring at the juncture of the sperm head and tail, have long been thought to act as diffusion barriers to help segregate important molecules required for fertilization within specific domains and to regulate migration of molecules between domains. In this investigation, we used a quantitative photobleaching technique (video-FRAP) to assess the efficacy of the posterior ring as a barrier to exchange of lipids between the postacrosomal and midpiece plasma membranes. A lipid reporter probe (1,1'-diduodecyl-3,3,3', 3'-tetramethylindocarbocyanine; DiIC(12)) was incorporated into the plasma membrane of live ram and boar spermatozoa, and the directionality of its diffusion across the posterior ring was measured by line-profile analysis. Results showed that DiIC(12) was able to traverse the posterior ring from the direction of the postacrosomal plasma membrane and to diffuse onto the midpiece plasma membrane. These results suggest that the posterior ring is not an immutable barrier to lipid exchange in mature spermatozoa and that there are other mechanisms for maintaining in-plane lipid asymmetry, such as differential phase behavior and interaction with the submembranous cytoskeleton.     

Ventilation and Its Effect on "Infinite Pool" Exchangers

SYNOPSIS. Unlike internal exchange surfaces, the skin contactsan "infinite pool" of air or water with which exchange of gases,water, ions, and other solutes may occur. Even though the "infinitepool" may be well mixed, an unstirred diffusion boundary layeris always present about the skin and may constitute a significantresistance to exchange. The thickness of the diffusion boundarylayer (as approximated by the fluid dynamic boundary layer)is related to the flow of the respiratory medium, viscosityand density of the medium, and the morphology of the exchangesurface. Oxygen microelectrode studies suggest that, in mostcircumstances, the diffusion boundary layer in water is at leastas thick as the blood-respiratory medium distance in amphibianskin. Accordingly, the movement of water about the skin {i.e.,skin ventilation) should have pronounced effects on cutaneousexchange, especially at low "free stream" velocities. Mountingphysiological evidence suggests that: (1) skin ventilation canaugment cutaneous gas exchange; and (2) some vertebrates activelyventilate their skins, especially in aquatic hypoxia. The ubiquityand significance of diffusion boundary layers are central toa general understanding of cutaneous exchange and all surface-mediatedexchange processes.     

Membrane adsorbers as purification tools for monoclonal antibody purification

Downstream purification processes for monoclonal antibody production typically involve multiple steps; some of them are conventionally performed by bead-based column chromatography. Affinity chromatography with Protein A is the most selective method for protein purification and is conventionally used for the initial capturing step to facilitate rapid volume reduction as well as separation of the antibody. However, conventional affinity chromatography has some limitations that are inherent with the method, it exhibits slow intraparticle diffusion and high pressure drop within the column. Membrane-based separation processes can be used in order to overcome these mass transfer limitations. The ligand is immobilized in the membrane pores and the convective flow brings the solute molecules very close to the ligand and hence minimizes the diffusional limitations associated with the beads. Nonetheless, the adoption of this technology has been slow because membrane chromatography has been limited by a lower binding capacity than that of conventional columns, even though the high flux advantages provided by membrane adsorbers would lead to higher productivity. This review considers the use of membrane adsorbers as an alternative technology for capture and polishing steps for the purification of monoclonal antibodies. Promising industrial applications as well as new trends in research will be addressed.     

Model of cell signal transduction in a three-dimensional domain

Intracellular signalling molecules form pathways inside the cell. These pathways carry a signal to target proteins which results in cellular responses. We consider a spherical cell with two internal compartments containing localized activating enzymes where as deactivating enzymes are spread uniformly through out the cytosol. Two diffusible signalling molecules are activated at the compartments and later deactivated in the cytosol due to deactivating enzymes. The two signalling molecules are a single link in a cascade reaction and form a self regulated dynamical system involving positive and negative feedback. Using matched asymptotic expansions we obtain approximate solutions of the steady state diffusion equation with a linear decay rate. We obtain three-dimensional concentration profiles for the signalling molecules. We also investigate an extension of the above system which has multiple cascade reactions occurring between multiple signalling molecules. Numerically, we show that the speed of the signal is an increasing function of the number of links in the cascade.     

Stochastic Simulation of Signal Transduction: Impact of the Cellular Architecture on Diffusion

The transduction of signals depends on the translocation of signaling molecules to specific targets. Undirected diffusion processes play a key role in the bridging of spaces between different cellular compartments. The diffusion of the molecules is, in turn, governed by the intracellular architecture. Molecular crowding and the cytoskeleton decrease macroscopic diffusion. This article shows the use of a stochastic simulation method to study the effects of the cytoskeleton structure on the mobility of macromolecules. Brownian dynamics and single particle tracking were used to simulate the diffusion process of individual molecules through a model cytoskeleton. The resulting average effective diffusion is in line with data obtained in the in vitro and in vivo experiments. It shows that the cytoskeleton structure strongly influences the diffusion of macromolecules. The simulation method used also allows the inclusion of reactions in order to model complete signaling pathways in their spatio-temporal dynamics, taking into account the effects of the cellular architecture.     

Extracellular space diffusion and extrasynaptic transmission

The diffusion of neuroactive substances in the extracellular space (ECS) plays an important role in short- and long-distance communication between nerve cells and is the underlying mechanism of extrasynaptic (volume) transmission. The diffusion properties of the ECS are described by three parameters: 1. ECS volume fraction alpha (alpha=ECS volume/total tissue volume), 2. tortuosity lambda (lambda2=free/apparent diffusion coefficient), reflecting the presence of diffusion barriers represented by, e.g., fine neuronal and glial processes or extracellular matrix molecules and 3. nonspecific uptake k'. These diffusion parameters differ in various brain regions, and diffusion in the CNS is therefore inhomogeneous. Moreover, diffusion barriers may channel the migration of molecules in the ECS, so that diffusion is facilitated in a certain direction, i.e. diffusion in certain brain regions is anisotropic. Changes in the diffusion parameters have been found in many physiological and pathological states in which cell swelling, glial remodeling and extracellular matrix changes are key factors influencing diffusion. Changes in ECS volume, tortuosity and anisotropy significantly affect the accumulation and diffusion of neuroactive substances in the CNS and thus extrasynaptic transmission, neuron-glia communication, transmitter "spillover" and synaptic cross-talk as well as cell migration, drug delivery and treatment.     

The variance of the distribution of traversal times in a capillary bed

A random walk model of capillary tracer transit times is developed that treats simulataneously: plug flow in the capillary, radial and axial diffusion in the capillary cylinder and tissue annulus, and endothelial barriers to solute transport. The mean transit time is simply the volume of distribution divided by blood flow. Variance of transit times has additive terms for radial, axial, and barrier influences that are reduceable to variances of simpler models of capillary exchange. The dependence of variance on the solute diffusion coefficient is not monotonic, but has a minimum near 0·5 × 10?6 cm²/s for reasonable parameters and no barrier, Small molecules like inert gases are expected to have larger variances with higher diffusion coefficients, while larger molecules and barrier limited solutes will have the reverse dependence. Available literature data indicates that capillary heterogeneity will have a major influence on whole-body variance of transit times.     

Particle Transport through Hydrogels Is Charge Asymmetric

Transport processes within biological polymer networks, including mucus and the extracellular matrix, play an important role in the human body, where they serve as a filter for the exchange of molecules and nanoparticles. Such polymer networks are complex and heterogeneous hydrogel environments that regulate diffusive processes through finely tuned particle-network interactions. In this work, we present experimental and theoretical studies to examine the role of electrostatics on the basic mechanisms governing the diffusion of charged probe molecules inside model polymer networks. Translational diffusion coefficients are determined by fluorescence correlation spectroscopy measurements for probe molecules in uncharged as well as cationic and anionic polymer solutions. We show that particle transport in the charged hydrogels is highly asymmetric, with diffusion slowed down much more by electrostatic attraction than by repulsion, and that the filtering capability of the gel is sensitive to the solution ionic strength. Brownian dynamics simulations of a simple model are used to examine key parameters, including interaction strength and interaction range within the model networks. Simulations, which are in quantitative agreement with our experiments, reveal the charge asymmetry to be due to the sticking of particles at the vertices of the oppositely charged polymer networks.     

Extrasynaptic transmission and the diffusion parameters of the extracellular space

Extrasynaptic volume transmission, mediated by the diffusion of neuroactive substances in the extracellular space (ECS), plays an important role in short- and long-distance communication between nerve cells. The ability of a substance to reach extrasynaptic high-affinity receptors via diffusion depends on the ECS diffusion parameters, ECS volume fraction alpha (alpha=ECS volume/total tissue volume) and tortuosity lambda (lambda2=free/apparent diffusion coefficient), which reflects the presence of diffusion barriers represented by, e.g., fine astrocytic processes or extracellular matrix molecules. These barriers channel the migration of molecules in the ECS, so that diffusion may be facilitated in a certain direction, i.e. anisotropic. The diffusion parameters alpha and lambda differ in various brain regions, and diffusion in the CNS is therefore inhomogeneous. Changes in diffusion parameters have been found in many physiological and pathological states, such as development and aging, neuronal activity, lactation, ischemia, brain injury, degenerative diseases, tumor growth and others, in which cell swelling, glial remodeling and extracellular matrix changes are key factors influencing diffusion. Changes in ECS volume, tortuosity and anisotropy significantly affect the accumulation and diffusion of neuroactive substances and thus extrasynaptic transmission, neuron-glia communication, mediator "spillover" and synaptic crosstalk as well as, cell migration. The various changes occurring during pathological states can be important for diagnosis, drug delivery and treatment.     

Numerical simulation of iontophoresis in the drug delivery system

The architecture and composition of stratum corneum act as barriers and limit the diffusion of most drug molecules and ions. Much effort has been made to overcome this barrier and it can be seen that iontophoresis has shown a good effect. Iontophoresis represents the application of low electrical potential to increase the transport of drugs into and across the skin or tissue. Iontophoresis is a noninvasive drug delivery system, and therefore, it is a useful alternative to drug transportation by injection. In this study, we present a numerical model and effects of electrical potential on the drug diffusion in the buccal tissue and the stratum corneum. The initial numerical results are in good comparison with experimental observation. We demonstrate that the application of an applied voltage can greatly improve the efficacy of localized drug delivery as compared to diffusion alone.     

Intracellular sorting and transport of proteins

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, integrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations.     

TRMP: a database of therapeutically relevant multiple pathways

SUMMARY: Disease processes often involve crosstalks between proteins in different pathways. Different proteins have been used as separate therapeutic targets for the same disease. Synergetic targeting of multiple targets has been explored in combination therapy of a number of diseases. Potential harmful interactions of multiple targeting have also been closely studied. To facilitate mechanistic study of drug actions and a more comprehensive understanding the relationship between different targets of the same disease, it is useful to develop a database of known therapeutically relevant multiple pathways (TRMPs). Information about non-target proteins and natural small molecules involved in these pathways also provides useful hint for searching new therapeutic targets and facilitate the understanding of how therapeutic targets interact with other molecules in performing specific tasks. The TRMPs database is designed to provide information about such multiple pathways along with related therapeutic targets, corresponding drugs/ligands, targeted disease conditions, constituent individual pathways, structural and functional information about each protein in the pathways. Cross links to other databases are also introduced to facilitate the access of information about individual pathways and proteins. AVAILABILITY: This database can be accessed at http://bidd.nus.edu.sg/group/trmp/trmp.asp and it currently contains 11 entries of multiple pathways, 97 entries of individual pathways, 120 targets covering 72 disease conditions together with 120 sets of drugs directed at each of these targets. Each entry can be retrieved through multiple methods including multiple pathway name, individual pathway name and disease name. SUPPLEMENTARY INFORMATION: http://bidd.nus.edu.sg/group/trmp/sm.pdf     

Neurotrophins act as neuroendocrine regulators of skin homeostasis in health and disease.

Neurotrophins regulate cutaneous innervation, act as growth and motility factors on structural skin cells such as keratinocytes and fibroblasts, modulate cutaneous immune function and even serve as stress mediators in skin biology. The multilayered neurotrophin interaction with skin biology through high affinity specific tyrosinekinase receptors and the Janus-faced p75 receptor, which depending on ligand and co-receptor expression can serve as a low-affinity pan-neurotrophin receptor or a high affinity proneurotrophin receptor, guaranties this neuroendocrine peptide family a central position in the control of skin homeostasis in health and disease. It is a challenging task for future research efforts to integrate our knowledge on differential neurotrophin expression patterns and signaling pathways into complex concepts of neuroendocrine tissue remodeling and pathogenetic processes. In addition, we need to improve our understanding of the role of neurotrophin processing enzymes, associated co-receptors and intracellular adaptor molecules in specific cutaneous cell populations to design precise interaction tools for research and treatment. Such tools will allow us to utilize this ancient growth factor family in the management of neurotrophin responsive pathogenetic pathways and cutaneous diseases such as neurogenic inflammation, peripheral nerve degeneration, wound healing, atopic dermatitis or psoriasis.     

Expression patterns of MDA-9/syntenin during development of the mouse embryo

Melanoma differentiation associated gene-9 (MDA-9)/syntenin is a PDZ domain-containing adaptor protein involved in multiple diverse cellular processes including organization of protein complexes in the plasma membrane, intracellular trafficking and cell surface targeting, synaptic transmission, and cancer metastasis. In the present study, we analyzed the expression pattern of MDA-9/syntenin during mouse development. MDA-9/syntenin was robustly expressed with tight regulation of its temporal and spatial expression during fetal development in the developing skin, spinal cord, heart, lung and liver, which are regulated by multiple signaling pathways in the process of organogenesis. Recent studies also indicate that MDA-9/syntenin is involved in the signaling pathways crucial during development such as Wnt, Notch and FGF. Taken together, these results suggest that MDA-9/syntenin may play a prominent role during normal mouse development in the context of cell proliferation as well as differentiation through modulating multiple signaling pathways as a crucial adaptor protein. Additionally, temporal regulation of MDA-9/syntenin expression may be required during specific stages and in specific tissues during development.     

Deviations from the flux ratio equation for chloride ions in ouabain- and acetazolamide-treated frog skin.

In order to establish whether or not chloride ions behave as freely moving particles in "passive", i.e. ouabain- and acetazolamide-treated, frog skin, tracer fluxes of 36Cl-have been measured while a voltage (generally +40 mV, serosal side positive) across the skin was applied. Ussing's flux ratio equation has been used as a criterion for this type of transport. One group of skin samples exhibited significant exchange diffusion phenomena. Most samples in a second group either behaved according to the flux ratio equation of showed significant and extreme exchange diffusion. From flux ratios obtained at two different voltages across various skin samples, showing extreme exchange diffusion, it appeared that the simple form of Kedem and Essig's law derived from irreversible thermodynamics, which is valid for homogeneous systems, does not apply to the type of exchange diffusion found. The system can, however, be described by a 1:1 exchange mechanism working in parallel with a diffusional pathway. The ratio exchange flux/observed efflux must then have a constant value (0.83) at the voltages appled, which implies that the exchange flux is voltage dependent. By comparison with iodide flux experiments as carried out by Ussing, it is shown that iodide exhibits the same type of exchange diffusion. A carrier, possibly responsible for the observed behaviour, is described.     

Critical Role of Tight Junctions in Drug Delivery across Epithelial and Endothelial Cell Layers

Epithelia in multicellular organisms constitute the frontier that separates the individual from the environment. Epithelia are sites of exchange as well as barriers, for the transit of ions and molecules from and into the organism. Therapeutic agents, in order to reach their target, frequently need to cross epithelial and endothelial sheets. Two routes are available for such purpose: the transcellular and the paracellular pathways. The former is employed by lipophilic drugs and by molecules selectively transported by channels, pumps and carriers present in the plasma membrane. Hydrophilic molecules cannot cross biological membranes, therefore their transepithelial transport could be significantly enhanced if they moved through the paracellular pathway. Transit through this route is regulated by tight junctions (TJs). The discovery in recent years of the molecular mechanisms of the TJ has allowed the design of different procedures to open the paracellular route in a reversible manner. These strategies could be used to enhance drug delivery across epithelial and endothelial barriers. The procedures employed include the use of peptides homologous to external loops of integral TJ proteins, silencing the expression of TJ proteins with antisense oligonucleotides and siRNAs as well as the use of toxins and proteins derived from microorganisms that target TJ proteins.     

Posttransition state desolvation of the hydrophobic core of the src-SH3 protein domain

The folding thermodynamics of the src-SH3 protein domain were characterized under refolding conditions through biased fully atomic molecular dynamics simulations with explicit solvent. The calculated free energy surfaces along several reaction coordinates revealed two barriers. The first, larger barrier was identified as the transition state barrier for folding, associated with the formation of the first hydrophobic sheet of the protein. phi values calculated from structures residing at the transition state barrier agree well with experimental phi values. The microscopic information obtained from our simulations allowed us to unambiguously assign intermediate phi values as the result of multiple folding pathways. The second, smaller barrier occurs later in the folding process and is associated with the cooperative expulsion of water molecules between the hydrophobic sheets of the protein. This posttransition state desolvation barrier cannot be observed through traditional folding experiments, but is found to be critical to the correct packing of the hydrophobic core in the final stages of folding. Hydrogen exchange and NMR experiments are suggested to probe this barrier.     

Deviations from the flux ratio equation for chloride ions in ouabain- and acetazolamide-treated frog skin

In order to establish whether or not chloride ions behave as freely moving particles in “passive”, i.e. ouabain-and acetazolamide-treated, frog skin, tracer fluxes of ³⁶Cl⁻ have been measured while a voltage (generally +40 mV, serosal side positive) across the skin was applied. Ussing's flux ratio equation has been used as a criterion for this type of transport. One group of skin samples exhibited significant exchange diffusion phenomena. Most samples in a second group either behaved according to the flux ratio equation or showed significant and extreme exchange diffusion. From flux ratios obtained at two different voltages across various skin samples, showing extreme exchange diffusion, it appeared that the simple form of Kedem and Essig's law derived from irreversible thermodynamics, which is valid for homogeneous systems, does not apply to the type of exchange diffusion found. The system can, however, be described by a 1 : 1 exchange mechanism working in parallel with a diffusional pathway. The ratio exchange flux/observed efflux must then have a constant value (0.83) at the voltages applied, which implies that the exchange flux is voltage dependent. By comparison with iodide flux experiments as carried out by Ussing, it is shown that iodide exhibits the same type of exchange diffusion. A carrier, possibly responsibe for the observed behaviour, is described.