Mechano-Regulation Of Meniscus Development And Maturation

The meniscus is an integral load bearing fibrous tissue of the knee joint that derives its mechanical function from the unique geometry and precise organization and composition of its extracellular matrix (ECM). While the importance of the highly specialized ECM is well appreciated in the mature meniscus, how this structural complexity is achieved during development remains poorly understood, and in particular, what interplay exists between the cells that build the matrix and their rapidly evolving microenvironment is unclear. To address these knowledge gaps, we begin by establishing a detailed timeline of the concurrent spatiotemporal changes that occur at both the cellular and matrix level during murine meniscus maturation, through use of Col1-YFP, Col2-CFP, Col10-mCherry fluorescent reporter mice, as well as histological analysis, and region specific high-throughput qPCR. We report that distinct cellular and matrix features defining specific meniscus tissue zones are present at birth, and that regional specialization continues during postnatal growth and maturation, possibly due to onset of load bearing use. Importantly, we define a framework for investigating the reciprocal feedback between cells and their evolving microenvironment—thus laying the foundation for future mechanistic work. Informed by the finding that key structural features of the meniscus matrix are established at birth, the remainder of this thesis addresses how this nascent organization is established. By analyzing key timepoints in knee joint development, we show that the genesis of ordered meniscus matrix is downstream of early cellular patterning characterized by marked fibrillation of the actin cytoskeleton. This suggests that cells and subcellular structures act as a physical template that directs alignment of the deposited fibrous matrix. Through the use of muscular dysgenesis (mdg) and splotch-delayed (Spd) mouse mutants that lack skeletal muscle contraction and joint motion, we further show that this critical cellular re-arrangement prior to meniscus formation does not fully occur without muscle contraction and leads to tissue dissociation—demonstrating that extrinsic forces play an instructive role in the tissue’s formation. Finally, we probe the impact of embryonic cell-mediated physical cues (adhesion, cytoskeletal arrangement) on subsequent meniscus assembly by generating targeted deletion of non-muscle myosin isoforms NM-IIA and NM-IIB (Myh dKO) in meniscus precursor cells during knee development. We demonstrate that cells of Myh dKO animals have defective cellular connectivity and so assemble a disorganized fibrillar matrix at birth, but these deficiencies in matrix alignment are somewhat corrected with postnatal maturation. Together, this work establishes that both cell-generated and extrinsic physical cues are imperative in the establishment of the initial meniscus structure that is built upon and further refined during postnatal growth

Kinetochore genes are coordinately up-regulated in human tumors as part of a FoxM1-related cell division program

The key player in directing proper chromosome segregation is the macromolecular kinetochore complex, which mediates DNA–microtubule interactions. Previous studies testing individual kinetochore genes documented examples of their overexpression in tumors relative to normal tissue, leading to proposals that up-regulation of specific kinetochore genes may promote tumor progression. However, kinetochore components do not function in isolation, and previous studies did not comprehensively compare the expression behavior of kinetochore components. Here we analyze the expression behavior of the full range of human kinetochore components in diverse published expression compendia, including normal tissues and tumor samples. Our results demonstrate that kinetochore genes are rarely overexpressed individually. Instead, we find that core kinetochore genes are coordinately regulated with other cell division genes under virtually all conditions. This expression pattern is strongly correlated with the expression of the forkhead transcription factor FoxM1, which binds to the majority of cell division promoters. These observations suggest that kinetochore gene up-regulation in cancer reflects a general activation of the cell division program and that altered expression of individual kinetochore genes is unlikely to play a causal role in tumorigenesis.Leukemia & Lymphoma Society of America (Scholar Award)National Institute of General Medical Sciences (U.S.) (Grant GM088313)American Cancer Society (Research Scholar Grant 121776)National Science Foundation (U.S.). Graduate Research Fellowshi

The CENP-L-N Complex Forms a Critical Node in an Integrated Meshwork of Interactions at the Centromere-Kinetochore Interface

During mitosis, the macromolecular kinetochore complex assembles on the centromere to orchestrate chromosome segregation. The properties and architecture of the 16-subunit Constitutive Centromere-Associated Network (CCAN) that allow it to build a robust platform for kinetochore assembly are poorly understood. Here, we use inducible CRISPR knockouts and biochemical reconstitutions to define the interactions between the human CCAN proteins. We find that the CCAN does not assemble as a linear hierarchy, and instead, each sub-complex requires multiple non-redundant interactions for its localization to centromeres and the structural integrity of the overall assembly. We demonstrate that the CENP-L-N complex plays a crucial role at the core of this assembly through interactions with CENP-C and CENP-H-I-K-M. Finally, we show that the CCAN is remodeled over the cell cycle such that sub-complexes depend on their interactions differentially. Thus, an interdependent meshwork within the CCAN underlies the centromere specificity and stability of the kinetochore.United States. National Institutes of Health (GM088313)United States. National Institutes of Health (GM108718)American Cancer Society (121776

Responsive Micromolds for Sequential Patterning of Hydrogel Microstructures

Microscale hydrogels have been shown to be beneficial for various applications such as tissue engineering and drug delivery. A key aspect in these applications is the spatial organization of biological entities or chemical compounds within hydrogel microstructures. For this purpose, sequentially patterned microgels can be used to spatially organize either living materials to mimic biological complexity or multiple chemicals to design functional microparticles for drug delivery. Photolithographic methods are the most common way to pattern microscale hydrogels but are limited to photocrosslinkable polymers. So far, conventional micromolding approaches use static molds to fabricate structures, limiting the resulting shapes that can be generated. Herein, we describe a dynamic micromolding technique to fabricate sequentially patterned hydrogel microstructures by exploiting the thermoresponsiveness of poly(N-isopropylacrylamide)-based micromolds. These responsive micromolds exhibited shape changes under temperature variations, facilitating the sequential molding of microgels at two different temperatures. We fabricated multicompartmental striped, cylindrical, and cubic microgels that encapsulated fluorescent polymer microspheres or different cell types. These responsive micromolds can be used to immobilize living materials or chemicals into sequentially patterned hydrogel microstructures which may potentially be useful for a range of applications at the interface of chemistry, materials science and engineering, and biology.United States. Army Research Office (Institute for Soldier Nanotechnologies at MIT, project DAAD-19-02-D-002)United States. Office of Naval ResearchNational Institutes of Health (U.S.) (DE013023)National Institutes of Health (U.S.) (DE016516)National Institutes of Health (U.S.) (HL092836)National Institutes of Health (U.S.) (DE019024)National Institutes of Health (U.S.) (EB012597)National Institutes of Health (U.S.) (AR057837)National Institutes of Health (U.S.) (DE021468)National Institutes of Health (U.S.) (HL099073

Responsive Microgrooves for the Formation of Harvestable Tissue Constructs

Given its biocompatibility, elasticity, and gas permeability, poly(dimethylsiloxane) (PDMS) is widely used to fabricate microgrooves and microfluidic devices for three-dimensional (3D) cell culture studies. However, conformal coating of complex PDMS devices prepared by standard microfabrication techniques with desired chemical functionality is challenging. This study describes the conformal coating of PDMS microgrooves with poly(N-isopropylacrylamide) (PNIPAAm) by using initiated chemical vapor deposition (iCVD). These microgrooves guided the formation of tissue constructs from NIH-3T3 fibroblasts that could be retrieved by the temperature-dependent swelling property and hydrophilicity change of the PNIPAAm. The thickness of swollen PNIPAAm films at 24 °C was approximately 3 times greater than at 37 °C. Furthermore, PNIPAAm-coated microgroove surfaces exhibit increased hydrophilicity at 24 °C (contact angle θ = 30° ± 2) compared to 37 °C (θ = 50° ± 1). Thus PNIPAAm film on the microgrooves exhibits responsive swelling with higher hydrophilicity at room temperature, which could be used to retrieve tissue constructs. The resulting tissue constructs were the same size as the grooves and could be used as modules in tissue fabrication. Given its ability to form and retrieve cell aggregates and its integration with standard microfabrication, PNIPAAm-coated PDMS templates may become useful for 3D cell culture applications in tissue engineering and drug discovery.Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (project DAAD-19-02-D-002)Charles Stark Draper LaboratoryNational Institutes of Health (U.S.) (DE01323)National Institutes of Health (U.S.) (DE016516)National Institutes of Health (U.S.) (HL092836)National Institutes of Health (U.S.) (DE019024)National Institutes of Health (U.S.) (EB007249)National Science Foundation (U.S.) (NSF Career Award (DMR0- 847287))United States. Office of Naval Research (Young Investigator Award)Wyss Institute for Biologically Inspired Engineerin