Escherichia coli condensin MukB stimulates topoisomerase IV activity by a direct physical interaction.

In contrast to the current state of knowledge in the field of eukaryotic chromosome segregation, relatively little is known about the mechanisms coordinating the appropriate segregation of bacterial chromosomes. In Escherichia coli, the MukB/E/F complex and topoisomerase IV (Topo IV) are both crucial players in this process. Topo IV removes DNA entanglements following the replication of the chromosome, whereas MukB, a member of the structural maintenance of chromosomes protein family, serves as a bacterial condensin. We demonstrate here a direct physical interaction between the dimerization domain of MukB and the C-terminal domain of the ParC subunit of Topo IV. In addition, we find that MukB alters the activity of Topo IV in vitro. Finally, we isolate a MukB mutant, D692A, that is deficient in its interaction with ParC and show that this mutant fails to rescue the temperature-sensitive growth phenotype of a mukB(-) strain. These results show that MukB and Topo IV are linked physically and functionally and indicate that the activities of these proteins are not limited to chromosome segregation but likely also play a key role in the control of higher-order bacterial chromosome structure.

Mechanical Memory Impairs Adipose-Derived Stem Cell (ASC) Adipogenic Capacity After Long-Term In Vitro Expansion.

INTRODUCTION: Adipose derived stem cells (ASCs) hold great promise for clinical applications such as soft tissue regeneration and for in vitro tissue models and are notably easy to derive in large numbers. Specifically, ASCs provide an advantage for in vitro models of adipose tissue, where they can be employed as tissue specific cells and for patient specific models. However, ASC in vitro expansion may unintentionally reduce adipogenic capacity due to the stiffness of tissue culture plastic (TCPS). METHODS: Here, we expanded freshly isolated ASCs on soft and stiff substrates for 4 passages before adipogenic differentiation. At the last passage we swapped the substrate from stiff to soft, or soft to stiff to determine if short term exposure to a different substrate altered adipogenic capacity. RESULTS: Expansion on stiff substrates reduced adipogenic capacity by 50% which was not rescued by swapping to a soft substrate for the last passage. Stiff substrates had greater nuclear area and gene expression of nesprin-2, a protein that mediates the tension of the nuclear envelope by tethering it to the actin cytoskeleton. Upon swapping to a soft substrate, the nuclear area was reduced but nesprin-2 levels did not fully recover, which differentially regulated cell commitment transcriptional factors. CONCLUSION: Therefore, in vitro expansion on stiff substrates must be carefully considered when the end-goal of the expansion is for adipose tissue or soft tissue applications.

Scaffold stiffness influences breast cancer cell invasion via EGFR-linked Mena upregulation and matrix remodeling.

Clinically, increased breast tumor stiffness is associated with metastasis and poorer outcomes. Yet, in vitro studies of tumor cells in 3D scaffolds have found decreased invasion in stiffer environments. To resolve this apparent contradiction, MDA-MB-231 breast tumor spheroids were embedded in 'low' (2 kPa) and 'high' (12 kPa) stiffness 3D hydrogels comprised of methacrylated gelatin/collagen I, a material that allows for physiologically-relevant changes in stiffness while matrix density is held constant. Cells in high stiffness materials exhibited delayed invasion, but more abundant actin-enriched protrusions, compared to those in low stiffness. We find that cells in high stiffness had increased expression of Mena, an invadopodia protein associated with metastasis in breast cancer, as a result of EGFR and PLCγ1 activation. As invadopodia promote invasion through matrix remodeling, we examined matrix organization and determined that spheroids in high stiffness displayed a large fibronectin halo. Interestingly, this halo did not result from increased fibronectin production, but rather from Mena/α5 integrin dependent organization. In high stiffness environments, FN1 knockout inhibited invasion while addition of exogenous cellular fibronectin lessened the invasion delay. Analysis of fibronectin isoforms demonstrated that EDA-fibronectin promoted invasion and that clinical invasive breast cancer specimens displayed elevated EDA-fibronectin. Combined, our data support a mechanism by which breast cancer cells respond to stiffness and render the environment conducive to invasion. More broadly, these findings provide important insight on the roles of matrix stiffness, composition, and organization in promoting tumor invasion.

Decoupling the effects of stiffness and fiber density on cellular behaviors via an interpenetrating network of gelatin-methacrylate and collagen.

The extracellular microenvironment provides critical cues that guide tissue development, homeostasis, and pathology. Deciphering the individual roles of these cues in tissue function necessitates the development of physically tunable culture platforms, but current approaches to create such materials have produced scaffolds that either exhibit a limited mechanical range or are unable to recapitulate the fibrous nature of in vivo tissues. Here we report a novel interpenetrating network (IPN) of gelatin-methacrylate (gelMA) and collagen I that enables independent tuning of fiber density and scaffold stiffness across a physiologically-relevant range of shear moduli (2-12 kPa), while maintaining constant extracellular matrix content. This biomaterial system was applied to examine how changes in the physical microenvironment affect cell types associated with the tumor microenvironment. By increasing fiber density while maintaining constant stiffness, we found that MDA-MB-231 breast tumor cells required the presence of fibers to invade the surrounding matrix, while endothelial cells (ECs) did not. Meanwhile, increasing IPN stiffness independently of fiber content yielded decreased invasion and sprouting for both MDA-MB-231 cells and ECs. These results highlight the importance of decoupling features of the microenvironment to uncover their individual effects on cell behavior, in addition to demonstrating that individual cell types within a tissue may be differentially affected by the same changes in physical features. The mechanical range and fibrous nature of this tunable biomaterial platform enable mimicry of a wide variety of tissues, and may yield more precise identification of targets which may be exploited to develop interventions to control tissue function.

Engineering approaches to study fibrosis in 3-D in vitro systems.

Fibrotic diseases occur in virtually every tissue of the body and are a major cause of mortality, yet they remain largely untreatable and poorly understood on a mechanistic level. The development of anti-fibrotic agents has been hampered, in part, by the insufficient fibrosis biomimicry provided by traditional in vitro platforms. This review focuses on recent advancements toward creating 3-D platforms that mimic key features of fibrosis, as well as the application of novel imaging and sensor techniques to analyze dynamic extracellular matrix remodeling. Several opportunities are highlighted to apply new tools from the fields of biomaterials, imaging, and systems biology to yield pathophysiologically relevant in vitro platforms that improve our understanding of fibrosis and may enable identification of potential treatment targets.

Conformational analysis of NMDA receptor GluN1, GluN2, and GluN3 ligand-binding domains reveals subtype-specific characteristics.

The NMDA receptor family of glutamate receptor ion channels is formed by obligate heteromeric assemblies of GluN1, GluN2, and GluN3 subunits. GluN1 and GluN3 bind glycine, whereas GluN2 binds glutamate. Crystal structures of the GluN1 and GluN3A ligand-binding domains (LBDs) in their apo states unexpectedly reveal open- and closed-cleft conformations, respectively, with water molecules filling the binding pockets. Computed conformational free energy landscapes for GluN1, GluN2A, and GluN3A LBDs reveal that the apo-state LBDs sample closed-cleft conformations, suggesting that their agonists bind via a conformational selection mechanism. By contrast, free energy landscapes for the AMPA receptor GluA2 LBD suggest binding of glutamate via an induced-fit mechanism. Principal component analysis reveals a rich spectrum of hinge bending, rocking, twisting, and sweeping motions that are different for the GluN1, GluN2A, GluN3A, and GluA2 LBDs. This variation highlights the structural complexity of signaling by glutamate receptor ion channels.

Analysis of high-affinity assembly for AMPA receptor amino-terminal domains.

Analytical ultracentrifugation (AUC) and steady-state fluorescence anisotropy were used to measure the equilibrium dissociation constant (Kd) for formation of dimers by the amino-terminal domains (ATDs) of the GluA2 and GluA3 subtypes of AMPA receptor. Previous reports on GluA2 dimerization differed in their estimate of the monomer-dimer Kd by a 2,400-fold range, with no consensus on whether the ATD forms tetramers in solution. We find by sedimentation velocity (SV) analysis performed using absorbance detection a narrow range of monomer-dimer Kd values for GluA2, from 5 to 11 nM for six independent experiments, with no detectable formation of tetramers and no effect of glycosylation or the polypeptide linker connecting the ATD and ligand-binding domains; for GluA3, the monomer-dimer Kd was 5.6 µM, again with no detectable tetramer formation. For sedimentation equilibrium (SE) experiments, a wide range of Kd values was obtained for GluA2, from 13 to 284 nM, whereas for GluA3, the Kd of 3.1 µM was less than twofold different from the SV value. Analysis of cell contents after the ∼1-week centrifuge run by silver-stained gels revealed low molecular weight GluA2 breakdown products. Simulated data for SE runs demonstrate that the apparent Kd for GluA2 varies with the extent of proteolysis, leading to artificially high Kd values. SV experiments with fluorescence detection for GluA2 labeled with 5,6-carboxyfluorescein, and fluorescence anisotropy measurements for GluA2 labeled with DyLight405, yielded Kd values of 5 and 11 nM, consistent with those from SV with absorbance detection. However, the sedimentation coefficients measured by AUC using absorbance and fluorescence systems were strikingly different, and for the latter are not consistent with hydrodynamic protein models. Thus, for unknown reasons, the concentration dependence of sedimentation coefficients obtained with fluorescence detection SV may be unreliable, limiting the usefulness of this technique for quantitative analysis.