Persistent tailoring of MSC activation through genetic priming.

Mesenchymal stem/stromal cells (MSCs) are an attractive platform for cell therapy due to their safety profile and unique ability to secrete broad arrays of immunomodulatory and regenerative molecules. Yet, MSCs are well known to require preconditioning or priming to boost their therapeutic efficacy. Current priming methods offer limited control over MSC activation, yield transient effects, and often induce expression of pro-inflammatory effectors that can potentiate immunogenicity. Here, we describe a 'genetic priming' method that can both selectively and sustainably boost MSC potency via the controlled expression of the inflammatory-stimulus-responsive transcription factor IRF1 (interferon response factor 1). MSCs engineered to hyper-express IRF1 recapitulate many core responses that are accessed by biochemical priming using the proinflammatory cytokine interferon-γ (IFNγ). This includes the upregulation of anti-inflammatory effector molecules and the potentiation of MSC capacities to suppress T cell activation. However, we show that IRF1-mediated genetic priming is much more persistent than biochemical priming and can circumvent IFNγ-dependent expression of immunogenic MHC class II molecules. Together, the ability to sustainably activate and selectively tailor MSC priming responses creates the possibility of programming MSC activation more comprehensively for therapeutic applications.

Membrane and Actin Tethering Transitions Help IQGAP1 Coordinate GTPase and Lipid Messenger Signaling.

The coordination of lipid messenger signaling with cytoskeletal regulation is central to many organelle-specific regulatory processes. This coupling often depends on the function of multidomain scaffolds that orchestrate transient interactions among multiple signaling intermediates and regulatory proteins on organelles. The number of possible scaffold interaction partners and the ability for these interactions to occur at different timescales makes investigations of scaffold functions challenging. This work employs live cell imaging to probe how the multidomain scaffold IQ motif containing GTPase activating protein 1 (IQGAP1) coordinates the activities of proteins affecting local actin polymerization, membrane processing, and phosphoinositide signaling. Using endosomes that are confined by a local actin network as a model system, we demonstrate that IQGAP1 can transition between different actin and endosomal membrane tethered states. Fast scaffold binding/disassociation transitions are shown to be driven by interactions between C-terminal scaffold domains and Rho GTPases at the membrane. Fluctuations in these binding modes are linked to negative regulation of actin polymerization. Although this control governs core elements of IQGAP1 dynamics, actin binding by the N-terminal calponin homology domain of the scaffold is shown to help the scaffold track the temporal development of endosome membrane markers, implying actin associations bolster membrane and actin coordination. Importantly, these effects are not easily distilled purely through standard (static) co-localization analyses or traditional pathway perturbations methods and were resolved by performing dynamic correlation and multiple regression analyses of IQGAP1 scaffold mutants. Using these capabilities with pharmacological inhibition, we provide evidence that membrane tethering is dependent on the activities of the lipid kinase phosphoinositide 3-kinase in addition to the Rho GTPases Rac1 and Cdc42. Overall, these methods and results point to a scaffold tethering mechanism that allows IQGAP1 to help control the amplitude of phosphoinositide lipid messenger signaling by coordinating signaling intermediate activities with the development and disassembly of local actin cytoskeletal networks.

Cryopreserved human umbilical cord versus acellular dermal matrix patches for in utero fetal spina bifida repair in a pregnant rat model.

OBJECTIVE: Despite significant improvement in spinal cord function after in utero spina bifida (SB) repair compared with traditional postnatal repair, over half of the children who undergo this procedure do not benefit completely. This lack of benefit has been attributed to closure methods of the defect, with subsequent spinal cord tethering at the repair site. Hence, a regenerative patch or material with antiinflammatory and anti-scarring properties may alleviate comorbidities with improved outcomes. The authors' primary objective was therefore to compare cryopreserved human umbilical cord (HUC) versus acellular dermal matrix (ADM) patches for regenerative repair of in utero SB lesions in an animal model. METHODS: In vivo studies were conducted in retinoic acid-induced SB defects in fetuses of Sprague-Dawley rats. HUC or ADM patches were sutured over the SB defects at a gestational age of 20 days. Repaired SB defect tissues were harvested after 48-52 hours. Tissue sections were immunofluorescently stained for the presence of neutrophils, macrophages, keratinocytes, meningeal cells, and astrocytes and for any associated apoptosis. In vitro meningeal or keratinocyte cell coculture experiments with the ADM and HUC patches were performed. All experiments were scored quantitatively in a blinded manner. RESULTS: Neutrophil counts and apoptotic cells were lower in the HUC-based repair group (n = 8) than in the ADM patch repair group (n = 7). In the HUC patch repair group, keratinocytes were present on the outer surface of the patch, meningeal cells were present on the inner surface of the patch adjacent to the neural placode, and astrocytes were noted to be absent. In the ADM patch repair group, all 3 cell types were present on both surfaces of the patch. In vitro studies showed that human meningeal cells grew preferentially on the mesenchymal side of the HUC patch, whereas keratinocytes showed tropism for the epithelial side, suggesting an inherent HUC-based cell polarity. In contrast, the ADM patch studies showed no polarity and decreased cellular infiltration. CONCLUSIONS: The HUC patch demonstrated reduced acute inflammation and apoptosis together with superior organization in regenerative cellular growth when compared with the ADM patch, and is therefore likely the better patch material for in utero SB defect repair. These properties may make the HUC biomaterial useful as a "meningeal patch" during spinal cord surgeries, thereby potentially reducing tethering and improving on spinal cord function.

Structural consequences of hereditary spastic paraplegia disease-related mutations in kinesin.

A wide range of mutations in the kinesin motor Kif5A have been linked to a neuronal disorder called hereditary spastic paraplegia (HSP). The position of these mutations can vary, and a range of different motile behaviors have been observed, indicating that the HSP mutants can alter distinct aspects of kinesin mechanochemistry. While focusing on four key HSP-associated mutants, this study examined the structural and dynamic perturbations that arise from these mutations using a series of different computational methods, ranging from bioinformatics analyses to all-atom simulations, that account for solvent effects explicitly. We show that two catalytic domain mutations (R280S and K253N) reduce the microtubule (MT) binding affinity of the kinesin head domains appreciably, while N256S has a much smaller impact. Bioinformatics analysis suggests that the stalk mutation A361V perturbs motor dimerization. Subsequent integration of these effects into a coarse-grained structure-based model of dimeric kinesin revealed that the order-disorder transition of the neck linker is substantially affected, indicating a hampered directionality and processivity of kinesin. The present analyses therefore suggest that, in addition to kinesin-MT binding and coiled-coil dimerization, HSP mutations affecting motor stepping transitions and processivity can lead to disease.

Molecular mechanisms of the interhead coordination by interhead tension in cytoplasmic dyneins.

Cytoplasmic dyneins play a major role in retrograde cellular transport by moving vesicles and organelles along microtubule filaments. Dyneins are multidomain motor proteins with two heads that coordinate their motion via their interhead tension. Compared with the leading head, the trailing head has a higher detachment rate from microtubules, facilitating the movement. However, the molecular mechanism of such coordination is unknown. To elucidate this mechanism, we performed molecular dynamics simulations on a cytoplasmic dynein with a structure-based coarse-grained model that probes the effect of the interhead tension on the structure. The tension creates a torque that influences the head rotating about its stalk. The conformation of the stalk switches from the α registry to the ß registry during the rotation, weakening the binding affinity to microtubules. The directions of the tension and the torque of the leading head are opposite to those of the trailing head, breaking the structural symmetry between the heads. The leading head transitions less often to the ß registry than the trailing head. The former thus has a greater binding affinity to the microtubule than the latter. We measured the moment arm of the torque from a dynein structure in the simulations to develop a phenomenological model that captures the influence of the head rotating about its stalk on the differential detachment rates of the two heads. Our study provides a consistent molecular picture for interhead coordination via interhead tension.

Molecular origin of the weak susceptibility of kinesin velocity to loads and its relation to the collective behavior of kinesins.

Motor proteins are active enzymatic molecules that support important cellular processes by transforming chemical energy into mechanical work. Although the structures and chemomechanical cycles of motor proteins have been extensively investigated, the sensitivity of a motor's velocity in response to a force is not well-understood. For kinesin, velocity is weakly influenced by a small to midrange external force (weak susceptibility) but is steeply reduced by a large force. Here, we utilize a structure-based molecular dynamic simulation to study the molecular origin of the weak susceptibility for a single kinesin. We show that the key step in controlling the velocity of a single kinesin under an external force is the ATP release from the microtubule-bound head. Only under large loading forces can the motor head release ATP at a fast rate, which significantly reduces the velocity of kinesin. It underpins the weak susceptibility that the velocity will not change at small to midrange forces. The molecular origin of this velocity reduction is that the neck linker of a kinesin only detaches from the motor head when pulled by a large force. This prompts the ATP binding site to adopt an open state, favoring ATP release and reducing the velocity. Furthermore, we show that two load-bearing kinesins are incapable of equally sharing the load unless they are very close to each other. As a consequence of the weak susceptibility, the trailing kinesin faces the challenge of catching up to the leading one, which accounts for experimentally observed weak cooperativity of kinesins motors.

The coordinating role of IQGAP1 in the regulation of local, endosome-specific actin networks.

IQGAP1 is a large, multi-domain scaffold that helps orchestrate cell signaling and cytoskeletal mechanics by controlling interactions among a spectrum of receptors, signaling intermediates, and cytoskeletal proteins. While this coordination is known to impact cell morphology, motility, cell adhesion, and vesicular traffic, among other functions, the spatiotemporal properties and regulatory mechanisms of IQGAP1 have not been fully resolved. Herein, we describe a series of super-resolution and live-cell imaging analyses that identified a role for IQGAP1 in the regulation of an actin cytoskeletal shell surrounding a novel membranous compartment that localizes selectively to the basal cortex of polarized epithelial cells (MCF-10A). We also show that IQGAP1 appears to both stabilize the actin coating and constrain its growth. Loss of compartmental IQGAP1 initiates a disassembly mechanism involving rapid and unconstrained actin polymerization around the compartment and dispersal of its vesicle contents. Together, these findings suggest IQGAP1 achieves this control by harnessing both stabilizing and antagonistic interactions with actin. They also demonstrate the utility of these compartments for image-based investigations of the spatial and temporal dynamics of IQGAP1 within endosome-specific actin networks.

Collective dynamics of processive cytoskeletal motors.

Major cellular processes are supported by various biomolecular motors that usually operate together as teams. We present an overview of the collective dynamics of processive cytokeletal motor proteins based on recent experimental and theoretical investigations. Experimental studies show that multiple motors function with different degrees of cooperativity, ranging from negative to positive. This effect depends on the mechanical properties of individual motors, the geometry of their connections, and the surrounding cellular environment. Theoretical models based on stochastic approaches underline the importance of intermolecular interactions, the properties of single motors, and couplings with cellular medium in predicting the collective dynamics. We discuss several features that specify the cooperativity in motor proteins. Based on this approach a general picture of collective dynamics of motor proteins is formulated, and the future directions and challenges are discussed.

Construction and analyses of elastically coupled multiple-motor systems.

Precision analyses of the collective motor behaviors have become important to dissecting mechanisms underlying the trafficking of subcellular commodities in eukaryotic cells. Here, we describe a synthetic approach to create structurally defined multiple protein complexes containing two elastically coupled motor molecules. Motors are connected using a simple DNA-scaffolding molecule and DNA-conjugated, artificial protein polymers that function as tunable elastic linkers. The procedure to self-assemble these components produces complexes in high synthetic yield and allows individual multiple-motor systems to be interrogated at the single-complex level. Methods to evaluate cooperative motor responses in a static optical trap are also discussed. While enabling the average transport properties of single/noninteracting and coupled motors to be compared, these procedures can provide insight into the extent to which motors cooperate productively via load sharing as well as the roles loading-rate-dependent phenomena play in collective motor functions.

Delineating cooperative responses of processive motors in living cells.

Characterizing the collective functions of cytoskeletal motors is critical to understanding mechanisms that regulate the internal organization of eukaryotic cells as well as the roles various transport defects play in human diseases. Though in vitro assays using synthetic motor complexes have generated important insights, dissecting collective motor functions within living cells still remains challenging. Here, we show that the protein heterodimerization switches FKBP-rapalog-FRB can be harnessed in engineered COS-7 cells to compare the collective responses of kinesin-1 and myosinVa motors to changes in motor number and cargo size. The dependence of cargo velocities, travel distances, and position noise on these parameters suggests that multiple myosinVa motors can cooperate more productively than collections of kinesins in COS-7 cells. In contrast to observations with kinesin-1 motors, the velocities and run lengths of peroxisomes driven by multiple myosinVa motors are found to increase with increasing motor density, but are relatively insensitive to the higher loads associated with transporting large peroxisomes in the viscoelastic environment of the COS-7 cell cytoplasm. Moreover, these distinctions appear to be derived from the different sensitivities of kinesin-1 and myosinVa velocities and detachment rates to forces at the single-motor level. The collective behaviors of certain processive motors, like myosinVa, may therefore be more readily tunable and have more substantial roles in intracellular transport regulatory mechanisms compared with those of other cytoskeletal motors.

Analysis of Cooperative Behavior in Multiple Kinesins Motor Protein Transport by Varying Structural and Chemical Properties.

Intracellular transport is a fundamental biological process during which cellular materials are driven by enzymatic molecules called motor proteins. Recent optical trapping experiments and theoretical analysis have uncovered many features of cargo transport by multiple kinesin motor protein molecules under applied loads. These studies suggest that kinesins cooperate negatively under typical transport conditions, although some productive cooperation could be achieved under higher applied loads. However, the microscopic origins of this complex behavior are still not well understood. Using a discrete-state stochastic approach we analyze factors that affect the cooperativity among kinesin motors during cargo transport. Kinesin cooperation is shown to be largely unaffected by the structural and mechanical parameters of a multiple motor complex connected to a cargo, but much more sensitive to biochemical parameters affecting motor-filament affinities. While such behavior suggests the net negative cooperative responses of kinesins will persist across a relatively wide range of cargo types, it is also shown that the rates with which cargo velocities relax in time upon force perturbations are influenced by structural factors that affect the free energies of and load distributions within a multiple kinesin complex. The implications of these later results on transport phenomena where loads change temporally, as in the case of bidirectional transport, are discussed.

Programming in situ immunofluorescence intensities through interchangeable reactions of dynamic DNA complexes.

The regulation of antibody reporting intensities is critical to various in situ fluorescence-imaging analyses. Although such control is often necessary to visualize sparse molecular targets, the ability to tune marker intensities is also essential for highly multiplexed imaging strategies in which marker reporting levels must be tuned both to optimize dynamic detection ranges and to minimize crosstalk between different signals. Existing chemical amplification approaches generally lack such control. Here, we demonstrate that linear and branched DNA complexes can be designed to function as interchangeable building blocks that can be assembled into organized, fluorescence-reporting complexes. We show that the ability to program DNA-strand-displacement reactions between these complexes offers new opportunities to deterministically tune the number of dyes that are coupled to individual antibodies in order both to increase and controllably balance marker reporting levels within fixed cells.

How the interplay between mechanical and nonmechanical interactions affects multiple kinesin dynamics.

Intracellular transport is supported by enzymes called motor proteins that are often coupled to the same cargo and function collectively. Recent experiments and theoretical advances have been able to explain certain behaviors of multiple motor systems by elucidating how unequal load sharing between coupled motors changes how they bind, step, and detach. However, nonmechanical interactions are typically overlooked despite several studies suggesting that microtubule-bound kinesins interact locally via short-range nonmechanical potentials. This work develops a new stochastic model to explore how these types of interactions influence multiple kinesin functions in addition to mechanical coupling. Nonmechanical interactions are assumed to affect kinesin mechanochemistry only when the motors are separated by less than three microtubule lattice sites, and it is shown that relatively weak interaction energies (~2 k(B)T) can have an appreciable influence over collective motor velocities and detachment rates. In agreement with optical trapping experiments on structurally defined kinesin complexes, the model predicts that these effects primarily occur when cargos are transported against loads exceeding single-kinesin stalling forces. Overall, these results highlight the interdependent nature of factors influencing collective motor functions, namely, that the way the bound configuration of a multiple motor system evolves under load determines how local nonmechanical interactions influence motor cooperation.

Collective dynamics of elastically coupled myosin V motors.

Characterization of the collective behaviors of different classes of processive motor proteins has become increasingly important to understand various intracellular trafficking and transport processes. This work examines the dynamics of structurally-defined motor complexes containing two myosin Va (myoVa) motors that are linked together via a molecular scaffold formed from a single duplex of DNA. Dynamic changes in the filament-bound configuration of these complexes due to motor binding, stepping, and detachment were monitored by tracking the positions of different color quantum dots that report the position of one head of each myoVa motor on actin. As in studies of multiple kinesins, the run lengths produced by two myosins are only slightly larger than those of single motor molecules. This suggests that internal strain within the complexes, due to asynchronous motor stepping and the resultant stretching of motor linkages, yields net negative cooperative behaviors. In contrast to multiple kinesins, multiple myosin complexes move with appreciably lower velocities than a single-myosin molecule. Although similar trends are predicted by a discrete state stochastic model of collective motor dynamics, these analyses also suggest that multiple myosin velocities and run lengths depend on both the compliance and the effective size of their cargo. Moreover, it is proposed that this unique collective behavior occurs because the large step size and relatively small stalling force of myoVa leads to a high sensitivity of motor stepping rates to strain.

Cooperative responses of multiple kinesins to variable and constant loads.

Microtubule-dependent transport is most often driven by collections of kinesins and dyneins that function in either a concerted fashion or antagonistically. Several lines of evidence suggest that cargo transport may not be influenced appreciably by the combined action of multiple kinesins. Yet, as in previous optical trapping experiments, the forces imposed on cargos will vary spatially and temporally in cells depending on a number of local environmental factors, and the influence of these conditions has been largely overlooked. Here, we characterize the dynamics of structurally defined complexes containing multiple kinesins under the controlled loads of an optical force clamp. While demonstrating that there are generic kinetic barriers that restrict the ability of multiple kinesins to cooperate productively, the spatial and temporal properties of applied loads is found to play an important role in the collective dynamics of multiple motor systems. We propose this dependence has implications for intracellular transport processes, especially for bidirectional transport.

Configuring robust DNA strand displacement reactions for in situ molecular analyses.

The number of distinct biomolecules that can be visualized within individual cells and tissue sections via fluorescence microscopy is limited by the spectral overlap of the fluorescent dye molecules that are coupled permanently to their targets. This issue prohibits characterization of important functional relationships between different molecular pathway components in cells. Yet, recent improved understandings of DNA strand displacement reactions now provides opportunities to create programmable labeling and detection approaches that operate through controlled transient interactions between different dynamic DNA complexes. We examined whether erasable molecular imaging probes could be created that harness this mechanism to couple and then remove fluorophore-bearing oligonucleotides to and from DNA-tagged protein markers within fixed cell samples. We show that the efficiency of marker erasing via strand displacement can be limited by non-toehold mediated stand exchange processes that lower the rates that fluorophore-bearing strands diffuse out of cells. Two probe constructions are described that avoid this problem and allow efficient fluorophore removal from their targets. With these modifications, we show one can at least double the number of proteins that can be visualized on the same cells via reiterative in situ labeling and erasing of markers on cells.