Absence Of Cargo Research Articles

Optimal Cargo Size for Active Diffusion of Biohybrid Microcarriers

As society paves its way towards device miniaturization and precision medicine, microscale actuation and transport become increasingly prominent research fields with high impact in both technological and clinical contexts. In order to accomplish movement of micron-sized objects towards specific target sites, active biohybrid transport systems, such as motile living cells that act as smart biochemically powered microcarriers, have been suggested as an alternative to synthetic microrobots. Inspired by the motility of leukocytes, we propose the amoeboid crawling of eukaryotic cells as a promising mechanism for transport of micron-sized cargoes and present an in-depth study of this type of composite active matter. Its transport properties result from the interactions of an active element (cell) and a passive one (cargo) and reveal an optimal cargo size that enhances the locomotion of the load-carrying cells, even exceeding their motility in the absence of cargo. The experimental findings are rationalized in terms of a biohybrid active particle model that describes the emergent cell-cargo dynamics and enables us to derive the long-time diffusive transport of amoeboid microcarriers. As amoeboid locomotion is commonly observed for mammalian cells such as leukocytes, our results lay the foundations for the study of transport performance of other medically relevant cell types and for extending our findings to more advanced transport tasks in complex environments, such as tissues.

Mast cell granule motility and exocytosis is driven by dynamic microtubule formation and kinesin-1 motor function.

Mast cells are tissue-resident immune cells that have numerous cytoplasmic granules which contain preformed pro-inflammatory mediators. Upon antigen stimulation, sensitized mast cells undergo profound changes to their morphology and rapidly release granule mediators by regulated exocytosis, also known as degranulation. We have previously shown that Rho GTPases regulate exocytosis, which suggests that cytoskeleton remodeling is involved in granule transport. Here, we used live-cell imaging to analyze cytoskeleton remodeling and granule transport in real-time as mast cells were antigen stimulated. We found that granule transport to the cell periphery was coordinated by de novo microtubule formation and not F-actin. Kinesore, a drug that activates the microtubule motor kinesin-1 in the absence of cargo, inhibited microtubule-granule association and significantly reduced exocytosis. Likewise, shRNA knock-down of Kif5b, the kinesin-1 heavy chain, also reduced exocytosis. Imaging showed granules accumulated in the perinuclear region after kinesore treatment or Kif5b knock-down. Complete microtubule depolymerization with nocodazole or colchicine resulted in the same effect. A biochemically enriched granule fraction showed kinesin-1 levels increase in antigen-stimulated cells, but are reduced by pre-treatment with kinesore. Kinesore had no effect on the levels of Slp3, a mast cell granule cargo adaptor, in the granule-enriched fraction which suggests that cargo adaptor recruitment to granules is independent of motor association. Taken together, these results show that granules associate with microtubules and are driven by kinesin-1 to facilitate exocytosis.

Structure and transport mechanism of P5B-ATPases

In human cells, P5B-ATPases execute the active export of physiologically important polyamines such as spermine from lysosomes to the cytosol, a function linked to a palette of disorders. Yet, the overall shape of P5B-ATPases and the mechanisms of polyamine recognition, uptake and transport remain elusive. Here we describe a series of cryo-electron microscopy structures of a yeast homolog of human ATP13A2-5, Ypk9, determined at resolutions reaching 3.4 Å, and depicting three separate transport cycle intermediates, including spermine-bound conformations. Surprisingly, in the absence of cargo, Ypk9 rests in a phosphorylated conformation auto-inhibited by the N-terminus. Spermine uptake is accomplished through an electronegative cleft lined by transmembrane segments 2, 4 and 6. Despite the dramatically different nature of the transported cargo, these findings pinpoint shared principles of transport and regulation among the evolutionary related P4-, P5A- and P5B-ATPases. The data also provide a framework for analysis of associated maladies, such as Parkinson’s disease.

Cargo competition for a dimerization interface restricts and stabilizes a bacterial protease adaptor

Bacterial protein degradation is a regulated process aided by protease adaptors that alter specificity of energy-dependent proteases. In Caulobacter crescentus, cell cycle-dependent protein degradation depends on a hierarchy of adaptors, such as the dimeric RcdA adaptor, which binds multiple cargo and delivers substrates to the ClpXP protease. RcdA itself is degraded in the absence of cargo, and how RcdA recognizes its targets is unknown. Here, we show that RcdA dimerization and cargo binding compete for a common interface. Cargo binding separates RcdA dimers, and a monomeric variant of RcdA fails to be degraded, suggesting that RcdA degradation is a result of self-delivery. Based on HDX-MS studies showing that different cargo rely on different regions of the dimerization interface, we generate RcdA variants that are selective for specific cargo and show cellular defects consistent with changes in selectivity. Finally, we show that masking of cargo binding by dimerization also limits substrate delivery to restrain overly prolific degradation. Using the same interface for dimerization and cargo binding offers an ability to limit excess protease adaptors by self-degradation while providing a capacity for binding a range of substrates.

Dynein Adaptors Such as Drosophila Bicaudal (Dm BicD) Recognize Cargo and are Required to Activate Dynein for Processive Transport

In the absence of cargo, auto-inhibited BicD cannot recruit dynein, but the underlying mechanism of cargo-induced activation is elusive. Our single-molecule processivity assays show that auto-inhibition is abolished in the full-length Bicaudal mutant F684I, which activates dynein motility even in the absence of cargo. To investigate the structural basis for activation, we determined the X-ray structure of the C-terminal cargo-binding domain (CTD) of Dm BicD-CTD/F684I at 2.4 Å resolution. The structure revealed that the mutant has a homotypic coiled-coil registry, in which the two helices of the homodimer are aligned, whereas the wild-type structure has an asymmetric registry, in which the two chains are vertically shifted by ∼1 helical turn in a portion of the coiled-coil. In the mutant, N- terminal ∼20-residue region is disordered for one of the two chains. MD simulation and CD spectroscopy data suggested the structural flexibility of N-terminal region of Dm BicD-CTD/F684I and confirmed the intact protein is in the crystal and alpha-helical. We propose that a coiled-coil registry shift activates BicD for dynein recruitment. The registry shift could either be induced by the F684I mutation or by cargo-binding. Also, the human homolog of the BicD-CTD/F684I mutant (BicD2-CTD/F743I) showed diminished binding to its cargo Nup358. Thus, we propose that in mammalia, a coiled-coil registry shift modulates cargo selection for BicD2-dependent transport pathways, which are important for brain development and chromosome segregation. Furthermore, we investigated the mechanism for cargo recognition by BicD2. Our data from Circular Dicroism spectroscopy and small-angle X-ray scattering suggest that the BicD2 binding domain of Nup358, which is intrinsically disordered on its own, takes on a helical structure when binding to BicD2. This alpha-helix of Nup358 could be a structural feature for cargo recognition by BicD2.

Removing the Polyanionic Cargo Requirement for Assembly of Alphavirus Core-Like Particles to Make an Empty Alphavirus Core.

The assembly of alphavirus nucleocapsid cores requires electrostatic interactions between the positively charged N-terminus of the capsid protein (CP) and the encapsidated polyanionic cargo. This system differs from many other viruses that can self-assemble particles in the absence of cargo, or form “empty” particles. We hypothesized that the introduction of a mutant, anionic CP could replace the need for charged cargo during assembly. In this work, we produced a CP mutant, Minus 38 (M38), where all N-terminal charged residues are negatively-charged. When wild-type (WT) and M38 CPs were mixed, they assembled into core-like particles (CLPs). These “empty” particles were of similar size and morphology to WT CLPs assembled with DNA cargo, but did not contain nucleic acid. When DNA cargo was added to the assembly mixture, the amount of M38 CP that was assembled into CLPs decreased, but was not fully excluded from the CLPs, suggesting that M38 competes with DNA to interact with WT CPs. The composition of CLPs can be tuned by altering the order of addition of M38 CP, WT CP, and DNA cargo. The ability to produce alphavirus CLPs that contain a range of amounts of encapsidated cargo, including none, introduces a new platform for packaging cargo for delivery or imaging purposes.

Coiled-coil registry shifts in the F684I mutant of Bicaudal D result in cargo-independent activation of dynein motility.

The dynein adaptor Drosophila Bicaudal D (BicD) is auto-inhibited and activates dynein motility only after cargo is bound, but the underlying mechanism is elusive. In contrast, we show that the full-length BicD/F684I mutant activates dynein processivity even in the absence of cargo. Our X-ray structure of the C-terminal domain of the BicD/F684I mutant reveals a coiled-coil registry shift; in the N-terminal region, the two helices of the homodimer are aligned, whereas they are vertically shifted in the wild-type. One chain is partially disordered and this structural flexibility is confirmed by computations, which reveal that the mutant transitions back and forth between the two registries. We propose that a coiled-coil registry shift upon cargo-binding activates BicD for dynein recruitment. Moreover, the human homolog BicD2/F743I exhibits diminished binding of cargo adaptor Nup358, implying that a coiled-coil registry shift may be a mechanism to modulate cargo selection for BicD2-dependent transport pathways.

European Web-Based Platform for Recording International Health Regulations Ship Sanitation Certificates: Results and Perspectives.

The purpose of this study was to report the data analysis results from the International Health Regulations (2005) Ship Sanitation Certificates (SSCs), recorded in the European Information System (EIS). International sea trade and population movements by ships can contribute to the global spread of diseases. SSCs are issued to ensure the implementation of control measures if a public health risk exists on board. EIS designed according to the World Health Organization (WHO) “Handbook for Inspection of Ships and Issuance of SSC”. Inspection data were recorded and SSCs issued by inspectors working at European ports were analysed. From July 2011–February 2017, 107 inspectors working at 54 ports in 11 countries inspected 5579 ships. Of these, there were 29 types under 85 flags (including 19 EU Member States flags). As per IHR (2005) 10,281 Ship Sanitation Control Exception Certificates (SSCECs) and 296 Ship Sanitation Control Certificates (SSCCs) were issued, 74 extensions to existing SSCs were given, 7565 inspection findings were recorded, and 47 inspections were recorded without issuing an SSC. The most frequent inspection findings were the lack of potable water quality monitoring reports (23%). Ships aged ≥12 years (odds ratio, OR = 1.77, 95% confidence intervals, CI = 1.37–2.29) with an absence of cargo at time of inspection (OR = 3.36, 95% CI = 2.51–4.50) had a higher probability of receiving an SSCC, while ships under the EU MS flag had a lower probability of having inspection findings (OR = 0.72, 95% CI = 0.66–0.79). Risk factors to prioritise the inspections according to IHR were identified by using the EIS. A global information system, or connection of national or regional information systems and data exchange, could help to better implement SSCs using common standards and procedures.

The role of the encapsulated cargo in microcompartment assembly.

Bacterial microcompartments are large, roughly icosahedral shells that assemble around enzymes and reactants involved in certain metabolic pathways in bacteria. Motivated by microcompartment assembly, we use coarse-grained computational and theoretical modeling to study the factors that control the size and morphology of a protein shell assembling around hundreds to thousands of molecules. We perform dynamical simulations of shell assembly in the presence and absence of cargo over a range of interaction strengths, subunit and cargo stoichiometries, and the shell spontaneous curvature. Depending on these parameters, we find that the presence of a cargo can either increase or decrease the size of a shell relative to its intrinsic spontaneous curvature, as seen in recent experiments. These features are controlled by a balance of kinetic and thermodynamic effects, and the shell size is assembly pathway dependent. We discuss implications of these results for synthetic biology efforts to target new enzymes to microcompartment interiors.

A small-molecule activator of kinesin-1 drives remodeling of the microtubule network

The microtubule motor kinesin-1 interacts via its cargo-binding domain with both microtubules and organelles, and hence plays an important role in controlling organelle transport and microtubule dynamics. In the absence of cargo, kinesin-1 is found in an autoinhibited conformation. The molecular basis of how cargo engagement affects the balance between kinesin-1's active and inactive conformations and roles in microtubule dynamics and organelle transport is not well understood. Here we describe the discovery of kinesore, a small molecule that in vitro inhibits kinesin-1 interactions with short linear peptide motifs found in organelle-specific cargo adaptors, yet activates kinesin-1's function of controlling microtubule dynamics in cells, demonstrating that these functions are mechanistically coupled. We establish a proof-of-concept that a microtubule motor-cargo interface and associated autoregulatory mechanism can be manipulated using a small molecule, and define a target for the modulation of microtubule dynamics.

Length of encapsidated cargo impacts stability and structure of in vitro assembled alphavirus core-like particles

In vitro assembly of alphavirus nucleocapsid cores, called core-like particles (CLPs), requires a polyanionic cargo. There are no sequence or structure requirements to encapsidate single-stranded nucleic acid cargo. In this work, we wanted to determine how the length of the cargo impacts the stability and structure of the assembled CLPs. We hypothesized that cargo neutralizes the basic region of the alphavirus capsid protein and if the cargo is long enough, it will also act to scaffold the CP monomers together. Experimentally we found that CLPs encapsidating short 27mer oligonucleotides were less stable than CLPs encapsidating 48mer or 90mer oligonucleotides under different chemical and thermal conditions. Furthermore, cryo-EM studies showed there were structural differences between CLPs assembled with 27mer and 48mer cargo. To mimic the role of the cargo in CLP assembly we made a mutant (4D) where we substituted a cluster of four Lys residues in the CP with four Asp residues. We found that these few amino acid substitutions were enough to initiate CLP assembly in the absence of cargo. The cargo-free 4D CLPs show higher resistance to ionic strength and increased temperature compared to wild-type cargo containing CLPs suggesting their CLP assembly mechanism might also be different.

Autoinhibition and cooperative activation mechanisms of cytoplasmic dynein.

Cytoplasmic dynein is a two-headed microtubule-based motor responsible for diverse intracellular movements, including minus-end-directed transport of organelles. The motility of cargo transporters is regulated according to the presence or absence of cargo; however, it remains unclear how cytoplasmic dynein achieves such regulation. Here, using a recombinant and native dynein complex in vitro, we show that lone, single dynein molecules are in an autoinhibited state, in which the two motor heads are stacked together. In this state, dynein moves diffusively along a microtubule with only a small bias towards the minus end of the microtubule. When the two heads were physically separated by a rigid rod, the movement of dynein molecules became directed and processive. Furthermore, assembly of multiple dynein molecules on a single cargo enabled them to move unidirectionally and generate force cooperatively. We thus propose a mechanism of autonomous on-off switching of cargo transport, in which single dynein molecules in the cell are autoinhibited through intramolecular head-head stacking and become active when they assemble as a team on a cargo.

Structural Insights into the Globular Tails of the Human Type V Myosins Myo5a, Myo5b, and Myo5c

Vertebrate type V myosins (MyoV) Myo5a, Myo5b, and Myo5c mediate transport of several different cargoes. All MyoV paralogs bind to cargo complexes mainly by their C-terminal globular domains. In absence of cargo, the globular domain of Myo5a inhibits its motor domain. Here, we report low-resolution SAXS models for the globular domains from human Myo5a, Myo5b, and Myo5c, which suggest very similar overall shapes of all three paralogs. We determined the crystal structures of globular domains from Myo5a and Myo5b, and provide a homology model for human Myo5c. When we docked the Myo5a crystal structure into a previously published electron microscopy density of the autoinhibited full-length Myo5a, only one domain orientation resulted in a good fit. This structural arrangement suggests the participation of additional region of the globular domain in autoinhibition. Quantification of the interaction of the Myo5a globular domain with its motor complex revealed a tight binding with dissociation half-life in the order of minutes, suggesting a rather slow transition between the active and inactive states.

Purification, crystallization and preliminary crystallographic analysis of the globular domain of the human type V myosin Myo5a.

Type V myosins constitute the main cargo-transporting class of myosin motors in higher eukaryotes. They are mainly defined by their C-terminal globular domain, which is required for cargo binding as well as for motor auto-inhibition in the absence of cargo. To date, high-resolution structures only exist for globular domains from yeast. Since the majority of cellular cargoes in yeast are very different from the cargoes in higher eukaryotes, structural insights into the domain organization of globular domains from human type V myosins are important. The globular domain of human Myo5a was cloned, expressed and crystallized and data sets were collected. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 75.04, b = 86.70, c = 131.41 Å, α = β = γ = 90°, and diffracted with data-collection quality to 2.5 Å resolution.

More than Just a Cargo Adapter: Melanophilin Prolongs Slow Processive Runs of Myosin Va

Melanophilin (Mlph) is a cargo adapter protein that links melanosomes via Rab27a to myosin Va (myoVa) for transport along actin. In the absence of cargo, full length myoVa is in equilibrium between a folded, inactive and an extended, active conformation. This equilibrium causes a deviation from a hand-over-hand stepping pattern due to altered gating between the heads (Armstrong et al. 2012). Cargo binding has been suggested to activate myoVa for transport. Here we used single molecule TIRF assays at near physiological ionic strength (150mM KCl) to determine the effect of Mlph on myoVa processivity. In the absence of Mlph, Qdot labeled full-length myoVa moved at a median velocity of 443nm/s, and showed the altered stepping pattern previously seen at lower ionic strength. Addition of Mlph recruited 14-times more motors to move processively, consistent with a simple model of cargo activation. The myoVa-Mlph complex also showed increased run lengths, with many motors traveling to the ends of the actin filament. In the presence of Mlph, myoVa moved much more slowly (median velocity=75nm/s). The speed distribution was asymmetrical and similar to speeds of melanosome movement observed in vivo. In the presence of Mlph, myoVa showed normal gating between the heads, and hand-over-hand steps on actin typical of a fully-active motor. Based on mutagenesis of Mlph the enhanced processivity depended on a positively charged cluster of amino acids in the actin binding site of Mlph. This suggests that Mlph acts as an electrostatic tether to limit myoVa dissociation from actin, a property likely to favor the transfer of melanosomes to adjacent keratinocytes in vivo. More generally, our results suggest that adapter proteins which link motors to cargo can affect motor properties in ways favorable for their biological role.

Cargo Activation of Full Length Myosin Va by Melanophilin Observed at the Single Molecule Level

Full-length myosin Va (myoVa) is auto-inhibited via a motor domain-globular tail interaction, unlike the truncated constitutively active myoVa-HMM. One potential mechanism to activate the full-length motor is cargo binding to the tail, which would compete with the head-tail interaction and trigger the molecule to extend and be activated for transport.In the absence of cargo, it was recently shown that full-length myoVa has two modes of interaction with actin in the presence of MgATP (Armstrong et al.). Most motors bind to actin but do not move, while the remainder show processive motion, but with a variable stepping pattern and altered gating. Here we investigate how binding of melanophilin (Mlph), which links the melanocyte-specific isoform of myoVa to the Rab27a(GTP)-melanosome complex, affects the properties of myoVa at the single-molecule level.In the absence of Mlph at 150mM KCl, a subset of Quantum dot labeled full-length myoVa moved at a median velocity of 566nm/s with the variable stepping pattern previously described, suggesting altered gating under these conditions. Addition of Mlph recruited 7-times more motors to move processively, consistent with a simple model of cargo activation. The myoVa-Mlph complex also showed increased run lengths, with many traveling to the ends of the actin filament. In the presence of Mlph, myoVa moved much more slowly (median velocity=76nm/s), leading to longer travel times on actin. When Mlph was bound to the motor the step sizes were normally distributed around 60 ± 14nm (SD) steps. Therefore, while myoVa moves more slowly along actin in the presence of the cargo adapter protein Mlph, it covers a greater distance with a more uniform and efficient stepping pattern. This slower processive movement could potentially facilitate binding of the Rab27a(GTP)-melanosome complex.

Autoinhibition of the kinesin-2 motor KIF17 via dual intramolecular mechanisms

Long-distance transport in cells is driven by kinesin and dynein motors that move along microtubule tracks. These motors must be tightly regulated to ensure the spatial and temporal fidelity of their transport events. Transport motors of the kinesin-1 and kinesin-3 families are regulated by autoinhibition, but little is known about the mechanisms that regulate kinesin-2 motors. We show that the homodimeric kinesin-2 motor KIF17 is kept in an inactive state in the absence of cargo. Autoinhibition is caused by a folded conformation that enables nonmotor regions to directly contact and inhibit the enzymatic activity of the motor domain. We define two molecular mechanisms that contribute to autoinhibition of KIF17. First, the C-terminal tail interferes with microtubule binding; and second, a coiled-coil segment blocks processive motility. The latter is a new mechanism for regulation of kinesin motors. This work supports the model that autoinhibition is a general mechanism for regulation of kinesin motors involved in intracellular trafficking events.

On the Movement of Cargo Driven by Molecular Motors and the Asymmetric Exclusion Processes

We consider the dynamics of particles driven by a collection of interacting molecular motors in the context of asymmetric simple exclusion processes (ASEP). The model is formulated to account for i) excluded volume interactions, ii) the observed asymmetry of the stochastic movement of individual motors and iii) interactions between motors and particles. Items (i) and (ii) form the basis of ASEP models and have already been considered in the literature to study the behavior of a collection of interacting motors in the absence of cargo. Item (iii) is new. It is introduced in this context as an attempt to describe explicitly the dependence of particle dynamics on the movement of motors [C.Goldman, E.Sena, Physica A 388(2009)]. The steady-state solutions to this model indicate that the system undergoes a phase transition of condensation type that can explain why, for sufficiently high motor densities, cargo velocity becomes independent of density in concert with the data obtained by Beeg et al [Biophys.J. 94(2008)]. The model predicts also that if more than one cargo-particle is present in the system these may exhibit inversion in movement direction. This means that the model suggests an alternative to explain the origin of the so called bidirectional movement of cargo according to which, inversions may happen in the presence of just one kind of motor.Financial support: Fundacao de Amparo a Pesquisa do Estado de Sao Paulo - FAPESP

Mammalian Kinesin-3 Motors Are Dimeric In Vivo and Move by Processive Motility upon Release of Autoinhibition

Kinesin-3 motors drive the transport of synaptic vesicles and other membrane-bound organelles in neuronal cells. In the absence of cargo, kinesin motors are kept inactive to prevent motility and ATP hydrolysis. Current models state that the Kinesin-3 motor KIF1A is monomeric in the inactive state and that activation results from concentration-driven dimerization on the cargo membrane. To test this model, we have examined the activity and dimerization state of KIF1A. Unexpectedly, we found that both native and expressed proteins are dimeric in the inactive state. Thus, KIF1A motors are not activated by cargo-induced dimerization. Rather, we show that KIF1A motors are autoinhibited by two distinct inhibitory mechanisms, suggesting a simple model for activation of dimeric KIF1A motors by cargo binding. Successive truncations result in monomeric and dimeric motors that can undergo one-dimensional diffusion along the microtubule lattice. However, only dimeric motors undergo ATP-dependent processive motility. Thus, KIF1A may be uniquely suited to use both diffuse and processive motility to drive long-distance transport in neuronal cells.

Regulated Conformation of Myosin V

We have found that myosin V, an important actin-based vesicle transporter, has a folded conformation that is coupled to inhibition of its enzymatic activity in the absence of cargo and Ca(2+). In the absence of Ca(2+) where the actin-activated MgATPase activity is low, purified brain myosin V sediments in the analytical ultracentrifuge at 14 S as opposed to 11 S in the presence of Ca(2+) where the activity is high. At high ionic strength it sediments at 10 S independent of Ca(2+), and its regulation is poor. These data are consistent with myosin V having a compact, inactive conformation in the absence of Ca(2+) and an extended conformation in the presence of Ca(2+) or high ionic strength. Electron microscopy reveals that in the absence of Ca(2+) the heads and tail are both folded to give a triangular shape, very different from the extended appearance of myosin V at high ionic strength. A recombinant myosin V heavy meromyosin fragment that is missing the distal portion of the tail domain is not regulated by calcium and has only a small change in sedimentation coefficient, which is in the opposite direction to that seen with intact myosin V. Electron microscopy shows that its heads are extended even in the absence of calcium. These data suggest that interaction between the motor and cargo binding domains may be a general mechanism for shutting down motor protein activity and thereby regulating the active movement of vesicles in cells.