Modeling Fusion/Fission‐Dependent Intracellular Transport of Fluid Phase Markers

A fundamental feature of eukaryotic cells is the presence of distinct membrane‐bound compartments having unique protein and lipid composition. These compartments are interconnected by active trafficking mechanisms that must direct macromolecules to defined locations, and at the same time maintain the protein and lipid composition of each organelle. It is well accepted that Rab proteins play a central role in intracellular transport regulating the recognition, fusion and fission of organelles. However, how the transport is achieved is not completely understood. We propose a model whereby a soluble component in the luminal compartment is transported along different Rab‐containing organelles that interact according to the following simple principles: (i) only organelles with the same or compatible Rab membrane domains can fuse; (ii) after fusion, an asymmetric fission occurs producing a tubule and a round‐shaped vesicle; and (iii) Rab membrane domains distribute asymmetrically between the two resulting organelles. When this model was tested in a simulation, efficient unidirectional transport was observed, while the compartment identity was preserved. All three principles were absolutely necessary for transport. The model is compatible with Rab association/dissociation dynamics and with Rab conversion. In simulations mimicking a simplified endocytic pathway, soluble and membrane‐associated markers were efficiently transported preserving the identity of the interacting compartments.     

Membrane identity and GTPase cascades regulated by toggle and cut‐out switches

Key cellular functions and developmental processes rely on cascades of GTPases. GTPases of the Rab family provide a molecular ID code to the generation, maintenance and transport of intracellular compartments. Here, we addressed the molecular design principles of endocytosis by focusing on the conversion of early endosomes into late endosomes, which entails replacement of Rab5 by Rab7. We modelled this process as a cascade of functional modules of interacting Rab GTPases. We demonstrate that intermodule interactions share similarities with the toggle switch described for the cell cycle. However, Rab5‐to‐Rab7 conversion is rather based on a newly characterized ‘cut‐out switch’ analogous to an electrical safety‐breaker. Both designs require cooperativity of auto‐activation loops when coupled to a large pool of cytoplasmic proteins. Live cell imaging and endosome tracking provide experimental support to the cut‐out switch in cargo progression and conversion of endosome identity along the degradative pathway. We propose that, by reconciling module performance with progression of activity, the cut‐out switch design could underlie the integration of modules in regulatory cascades from a broad range of biological processes.     

Human ARF4 expression rescues sec7 mutant yeast cells.

Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events. The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae. To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants. We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature. The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells. Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function. Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic. In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner. This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics.     

Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin

The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin‐interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target‐search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance.     

Analytical aspects of the Brownian motor effect in randomly flashing ratchets

The muscle contraction, operation of ATP synthase, maintaining the shape of a cell are believed to be secured by motor proteins, which can be modelled using the Brownian ratchet mechanism. We consider the randomly flashing ratchet model of a Brownian motor, where the particles can be in two states, only one of which is sensitive the applied spatially periodic potential (the mathematical setting is a pair of weakly coupled reaction-diffusion and Fokker–Planck equations). We prove that this mechanism indeed generates unidirectional transport by showing that the amount of mass in the wells of the potential decreases/increases from left to right. The direction of transport is unambiguously determined by the location of each minimum of the potential with respect to the so-called diffusive mean of its adjacent maxima. The transport can be generated not only by an asymmetric potential, but also by a symmetric potential and asymmetric transition rates, and as a consequence of the general result we derive explicit conditions when the latter happens. When the transitions are localized on narrow active sites in the protein conformation space, we find a more explicit characterization of the bulk transport direction, and infer that some common preconditions of the motor effect are redundant.     

The switching dynamics of the bacterial flagellar motor

Many bacteria are propelled by flagellar motors that stochastically switch between the clockwise and counterclockwise rotation direction. Although the switching dynamics is one of their most important characteristics, the mechanisms that control it are poorly understood. We present a statistical–mechanical model of the bacterial flagellar motor. At its heart is the assumption that the rotor protein complex, which is connected to the flagellum, can exist in two conformational states and that switching between these states depends on the interactions with the stator proteins, which drive the rotor. This couples switching to rotation, making the switch sensitive to torque and speed. Another key element is that after a switch, it takes time for the load to build up, due to conformational transitions of the flagellum. This slow relaxation dynamics of the filament leads, in combination with the load dependence of the switching frequency, to a characteristic switching time, as recently observed. Hence, our model predicts that the switching dynamics is not only controlled by the chemotaxis‐signaling network, but also by mechanical feedback of the flagellum.     

A simple guide to biochemical approaches for analyzing protein-lipid interactions

Eukaryotic cells contain many different membrane compartments with characteristic shapes, lipid compositions, and dynamics. A large fraction of cytoplasmic proteins associate with these membrane compartments. Such protein-lipid interactions, which regulate the subcellular localizations and activities of peripheral membrane proteins, are fundamentally important for a variety of cell biological processes ranging from cytoskeletal dynamics and membrane trafficking to intracellular signaling. Reciprocally, many membrane-associated proteins can modulate the shape, lipid composition, and dynamics of cellular membranes. Determining the exact mechanisms by which these proteins interact with membranes will be essential to understanding their biological functions. In this Technical Perspective, we provide a brief introduction to selected biochemical methods that can be applied to study protein-lipid interactions. We also discuss how important it is to choose proper lipid composition, type of model membrane, and biochemical assay to obtain reliable and informative data from the lipid-interaction mechanism of a protein of interest.     

A possible mechanism for self-coordination of bidirectional traffic across nuclear pores

Nuclear pore complexes are constantly confronted by large fluxes of macromolecules and macromolecular complexes that need to get into and out of the nucleus. Such bidirectional traffic occurring in a narrow channel can easily lead to jamming. How then is passage between the nucleus and cytoplasm maintained under the varying conditions that arise during the lifetime of the cell? Here, we address this question using computer simulations in which the behaviour of the ensemble of transporting cargoes is analysed under different conditions. We suggest that traffic can exist in two distinct modes, depending on the concentration of cargoes and dissociation rates of the transport receptor-cargo complexes from the pores. In one mode, which prevails when dissociation is quick and cargo concentration is low, transport in either direction proceeds uninterrupted by transport in the other direction. The result is that the overall traffic direction fluctuates rapidly and unsystematically between import and export. Remarkably, when cargo concentrations are high and disassociation is slow, another mode takes over in which traffic proceeds in one direction for a certain extent of time, after which it flips direction for another period. The switch between this, more regulated, mode of transport and the other, quickly fluctuating state, does not require an active gating mechanism but rather occurs spontaneously through the dynamics of the transported particles themselves. The determining factor for the behaviour of traffic is found to be the exit rate from the pore channel, which is directly related to the activity of the Ran system that controls the loading and release of cargo in the appropriate cellular compartment.     

Revealing A-Raf functions through its interactome

A-Raf is a member of the Raf kinase family. Unlike B-Raf and C-Raf, the functions of A-Raf remain obscure. To gain more insight into the biological functions of A-Raf, we investigated the A-Raf interactome using proteomics. We found 132 proteins that interact with A-Raf and confirmed the interaction of 12 of these proteins with A-Raf by western blotting. Our data suggested that A-Raf regulates apoptosis, RNA catabolism, GTPase activity, and cell adhesion by interacting with proteins located in different cellular compartments. We identified all ten hallmarks of cancer in these interacting proteins, suggesting that A-Raf is involved in carcinogenesis. Our results also indicated that A-Raf may play a role in different diseases and signaling pathways. These findings have identified potential regulators of A-Raf and provide a systemic insight into its biological functions.     

Calcium tips the balance: a microtubule plus end to lattice binding switch operates in the carboxyl terminus of BPAG1n4

Microtubules (MTs) are integral to numerous cellular functions, such as cell adhesion, differentiation and intracellular transport. Their dynamics are largely controlled by diverse MT‐interacting proteins, but the signalling mechanisms that regulate these interactions remain elusive. In this report, we identify a rapid, calcium‐regulated switch between MT plus end interaction and lattice binding within the carboxyl terminus of BPAG1n4. This switch is EF‐hand dependent, and mutations of the EF‐hands abolish this dynamic behaviour. Our study thus uncovers a new, calcium‐dependent regulatory mechanism for a spectraplakin, BPAG1n4, at the MT plus end.     

Intracellular trafficking of Niemann-Pick C proteins 1 and 2: obligate components of subcellular lipid transport

Niemann-Pick C 1 (NPC1) is a large integral membrane glycoprotein that resides in late endosomes, whereas NPC2 is a small soluble protein found in the lumen of lysosomes. Mutations in either NPC1 or NPC2 result in aberrant lipid transport from endocytic compartments, which results in lysosomal storage of a complex mixture of lipids, primarily cholesterol and glycosphingolipids. What are the biological functions of the NPC1 and NPC2 proteins? Here we review what is known about the intracellular itinerary of these two proteins as they facilitate lipid transport. We propose that the intracellular trafficking patterns of these proteins will provide clues about their function.     

Aquaporin Trafficking in Plant Cells: An Emerging Membrane‐Protein Model

Aquaporins (AQPs) are channel proteins that facilitate the transport of water and small solutes across biological membranes. In plants, AQPs exhibit a high multiplicity of isoforms in relation to a high diversity of sub‐cellular localizations, at the plasma membrane (PM) and in various intracellular compartments. Some members also exhibit a dual localization in distinct cell compartments, whereas others show polarized or domain‐specific expression at the PM or tonoplast, respectively. A diversity of mechanisms controlling the routing of newly synthesized AQPs towards their destination membranes and involving diacidic motifs, phosphorylation or tetramer assembly is being uncovered. Recent approaches using single particle tracking, fluorescence correlation spectroscopy and fluorescence recovery after photobleaching have, in combination with pharmacological interference, stressed the peculiarities of AQP sub‐cellular dynamics in environmentally challenging conditions. A role for clathrin and sterol‐rich domains in cell surface dynamics and endocytosis of PM AQPs was uncovered. These recent advances provide deep insights into the cellular mechanisms of water transport regulation in plants. They also point to AQPs as an emerging model for studying the sub‐cellular dynamics of plant membrane proteins .     

Characterization of protein hubs by inferring interacting motifs from protein interactions

The characterization of protein interactions is essential for understanding biological systems. While genome-scale methods are available for identifying interacting proteins, they do not pinpoint the interacting motifs (e.g., a domain, sequence segments, a binding site, or a set of residues). Here, we develop and apply a method for delineating the interacting motifs of hub proteins (i.e., highly connected proteins). The method relies on the observation that proteins with common interaction partners tend to interact with these partners through a common interacting motif. The sole input for the method are binary protein interactions; neither sequence nor structure information is needed. The approach is evaluated by comparing the inferred interacting motifs with domain families defined for 368 proteins in the Structural Classification of Proteins (SCOP). The positive predictive value of the method for detecting proteins with common SCOP families is 75% at sensitivity of 10%. Most of the inferred interacting motifs were significantly associated with sequence patterns, which could be responsible for the common interactions. We find that yeast hubs with multiple interacting motifs are more likely to be essential than hubs with one or two interacting motifs, thus rationalizing the previously observed correlation between essentiality and the number of interacting partners of a protein. We also find that yeast hubs with multiple interacting motifs evolve slower than the average protein, contrary to the hubs with one or two interacting motifs. The proposed method will help us discover unknown interacting motifs and provide biological insights about protein hubs and their roles in interaction networks.     

A network model of early events in epidermal growth factor receptor signaling that accounts for combinatorial complexity

We consider a model of early events in signaling by the epidermal growth factor (EGF) receptor (EGFR). The model includes EGF, EGFR, the adapter proteins Grb2 and Shc, and the guanine nucleotide exchange factor Sos, which is activated through EGF-induced formation of EGFR-Grb2-Sos and EGFR-Shc-Grb2-Sos assemblies at the plasma membrane. The protein interactions involved in signaling can potentially generate a diversity of protein complexes and phosphoforms; however, this diversity has been largely ignored in models of EGFR signaling. Here, we develop a model that accounts more fully for potential molecular diversity by specifying rules for protein interactions and then using these rules to generate a reaction network that includes all chemical species and reactions implied by the protein interactions. We obtain a model that predicts the dynamics of 356 molecular species, which are connected through 3749 unidirectional reactions. This network model is compared with a previously developed model that includes only 18 chemical species but incorporates the same scope of protein interactions. The predictions of this model are reproduced by the network model, which also yields new predictions. For example, the network model predicts distinct temporal patterns of autophosphorylation for different tyrosine residues of EGFR. A comparison of the two models suggests experiments that could lead to mechanistic insights about competition among adapter proteins for EGFR binding sites and the role of EGFR monomers in signal transduction.     

Evolution of dispersal in open advective environments

We consider a two-species competition model in a one-dimensional advective environment, where individuals are exposed to unidirectional flow. The two species follow the same population dynamics but have different random dispersal rates and are subject to a net loss of individuals from the habitat at the downstream end. In the case of non-advective environments, it is well known that lower diffusion rates are favored by selection in spatially varying but temporally constant environments, with or without net loss at the boundary. We consider several different biological scenarios that give rise to different boundary conditions, in particular hostile and “free-flow” conditions. We establish the existence of a critical advection speed for the persistence of a single species. We derive a formula for the invasion exponent and perform a linear stability analysis of the semi-trivial steady state under free-flow boundary conditions for constant and linear growth rate. For homogeneous advective environments with free-flow boundary conditions, we show that populations with higher dispersal rate will always displace populations with slower dispersal rate. In contrast, our analysis of a spatially implicit model suggest that for hostile boundary conditions, there is a unique dispersal rate that is evolutionarily stable. Nevertheless, both scenarios show that unidirectional flow can put slow dispersers at a disadvantage and higher dispersal rate can evolve.     

Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP)

Rab GTPases recruit effector proteins, via their GTP-dependent switch regions, to distinct subcellular compartments. Rab11 and Rab25 are closely related small GTPases that bind to common effectors termed the Rab11 family of interacting proteins (FIPs). The FIPs are organized into two subclasses (class I and class II) based on sequence and domain organization, and both subclasses contain a highly conserved Rab-binding domain at their C termini. Yeast two-hybrid and biochemical studies have revealed that the more distantly related Rab14 also interacts with class I FIPs. Here, we perform detailed structural, thermodynamic, and cellular analyses of the interactions between Rab14 and one of the class I FIPs, the Rab-coupling protein (RCP), to clarify the molecular aspects of the interaction. We find that Rab14 indeed binds to RCP, albeit with reduced affinity relative to conventional Rab11-FIP and Rab25-FIP complexes. However, in vivo, Rab11 recruits RCP onto biological membranes. Furthermore, biophysical analyses reveal a noncanonical 1:2 stoichiometry between Rab14-RCP in dilute solutions, in contrast to Rab11/25 complexes. The structure of Rab14-RCP reveals that Rab14 interacts with the canonical Rab-binding domain and also provides insight into the unusual properties of the complex. Finally, we show that both the Rab coupling protein and Rab14 function in neuritogenesis.     

Expression analysis and chromosomal assignment of PRA1 and RILP genes

PRA1 (prenylated Rab acceptor) is a general regulator of Rab proteins, while RILP (Rab interacting lysosomal protein) is a specific effector for Rab7. It has been shown that PRA1 interacts with Rab proteins and with VAMP2. Therefore PRA1 is probably an important factor for membrane traffic, linking together the function of Rab proteins and SNAREs. RILP has a key role in the control of transport to degradative compartments together with Rab7 and probably links Rab7 function to the cytoskeleton. Here we have studied by Northern blot the expression of the two genes in several different human tissues. The 0.8-kb mRNA for human PRA1 is ubiquitously expressed, while the two mRNAs for RILP are differentially expressed. In addition, we have assigned the human PRA1 gene to chromosome 19q13.13-q13.2 and the human RILP gene to chromosome 17p13.3.     

EGGTART: A tool to visualize the dynamics of biophysical transport under the inhomogeneous l-TASEP

The totally asymmetric simple exclusion process (TASEP), which describes the stochastic dynamics of interacting particles on a lattice, has been actively studied over the past several decades and applied to model important biological transport processes. Here, we present a software package, called EGGTART (Extensive GUI gives TASEP-realization in Real Time), which quantifies and visualizes the dynamics associated with a generalized version of the TASEP with an extended particle size and heterogeneous jump rates. This computational tool is based on analytic formulas obtained from deriving and solving the hydrodynamic limit of the process. It allows an immediate quantification of the particle density, flux, and phase diagram, as a function of a few key parameters associated with the system, which would be difficult to achieve via conventional stochastic simulations. Our software should therefore be of interest to biophysicists studying general transport processes and can in particular be used in the context of gene expression to model and quantify mRNA translation of different coding sequences.