SUMMARYAlong with Parkin, PINK1 plays a critical role in maintaining mitochondrial quality control. Although PINK1 is expressed constitutively, its level is kept low in healthy mitochondria by polyubiquitination and ensuing proteasomal degradation of its mature, 52 kDa, form. We show here that the target of PINK1 polyubiquitination is the mature form and is mediated by ubiquitination of a conserved lysine at position 137. Notably, the full-length protein also contains Lys-137 but is not ubiquitinated. On the basis of our data, we propose that cleavage of full-length PINK1 at Phe-104 disrupts the major hydrophobic membrane-spanning domain in the protein, inducing a conformation change in the resultant mature form that exposes Lys-137 to the cytosol for subsequent modification by the ubiquitination machinery. Thus, the balance between the full-length and mature PINK1 allows its levels to be regulated via ubiquitination of the mature form and ensures that PINK1 functions as a mitochondrial quality control factor.

Maintaining a pool of functional mitochondria requires degradation of damaged ones within the cell. PINK1 is critical in this qualitycontrol process: loss of mitochondrial membrane potential causes PINK1 to accumulate on the mitochondrial surface, triggering mitophagy. However, little is known about how PINK1 is regulated. Recently, we showed that PINK1 content is kept low in healthy mitochondria by continuous ubiquitination and proteasomal degradation of its mature form via a mechanism inconsistent with the proposed N-end rule process. Using both human female and monkey cell lines, we now demonstrate that once generated within the mitochondria, 52 kDa PINK1 adopts a mitochondrial topology most consistent with it being at the mitochondrial-endoplasmic reticulum (ER) interface. From this particular submitochondrial location, PINK1 interacts with components of the ER-associated degradation pathway, such as the E3 ligases gp78 and HRD1, which cooperate to catalyze PINK1 ubiquitination. The valosin-containing protein and its cofactor, UFD1, then target ubiquitinated PINK1 for proteasomal degradation. Our data show that PINK1 in healthy mitochondria is negatively regulated via an interplay between mitochondria and ER, and shed light on how this mitochondrial protein gains access to the proteasome.

Cells embedded in the extracellular matrix of tissues play a critical role in maintaining homeostasis while promoting integration and regeneration following damage or disease. Emerging engineered biomaterials utilize decellularized extracellular matrix as a tissue-specific support structure; however, many dense, structured biomaterials unfortunately demonstrate limited formability, fail to promote cell migration, and result in limited tissue repair. Here, a reinforced composite material of densely packed acellular extracellular matrix microparticles in a hydrogel, termed tissue clay, that can be molded and crosslinked to mimic native tissue architecture is developed. Hyaluronic acid-based hydrogels are utilized, amorphously packed with acellular cartilage tissue particulated to ≈125-250 microns in diameter and defined a percolation threshold of 0.57 (v/v) beyond which the compressive modulus exceeded 300 kPa. Remarkably, primary chondrocytes recellularize particles within 48 h, a process driven by chemotaxis, exhibit distributed cellularity in large engineered composites, and express genes consistent with native cartilage repair. In addition, broad utility of tissue clays through recellularization and persistence of muscle, skin, and cartilage composites in an in vivo mouse model is demonstrated. The findings suggest optimal material architectures to balance concurrent demands for large-scale mechanical properties while also supporting recellularization and integration of dense musculoskeletal and connective tissues.

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Cells embedded in the extracellular matrix of tissues play a critical role in maintaining homeostasis while promoting integration and regeneration following damage or disease. Emerging engineered biomaterials utilize decellularized extracellular matrix as a tissue-specific support structure; however, many dense, structured biomaterials unfortunately demonstrate limited formability, fail to promote cell migration, and result in limited tissue repair. Here, we developed a reinforced composite material of densely packed acellular extracellular matrix microparticles in a hydrogel, termed tissue clay, that can be molded and crosslinked to mimic native tissue architecture. We utilized hyaluronic acid-based hydrogels, amorphously packed with acellular articular cartilage tissue particulated to ~125-250 microns in diameter and defined a percolation threshold of 0.57 (v/v) beyond which the compressive modulus exceeded 300kPa. Remarkably, primary chondrocytes recellularized particles within 48 hours, a process driven by chemotaxis, exhibited distributed cellularity in large engineered composites, and expressed genes consistent with native cartilage repair. We additionally demonstrated broad utility of tissue clays through recellularization and persistence of muscle, skin, and cartilage composites in a subcutaneous in vivo mouse model. Our findings suggest optimal strategies and material architectures to balance concurrent demands for large-scale mechanical properties while also supporting integration of dense musculoskeletal and connective tissues.

Dedifferentiation of chondrocytes during in vitro passaging before implantation, and post implantation in vivo, is a critical limitation in cartilage tissue engineering. Several biophysical features define the dedifferentiated state including a flattened cell morphology and increased stress fiber formation. However, how dedifferentiation influences nuclear mechanics, and the possible long-term implications of this state, are unknown. In this study, we investigated how chondrocyte dedifferentiation affects the mechanics of the chromatin architecture inside the cell nucleus and the gene expression of the structural proteins located at the nuclear envelope. Through an experimental model of cell stretching and a detailed spatial intranuclear strain quantification, we identified that strain is amplified and distribution of strain within the chromatin is altered under tensile loading in the dedifferentiated state. Further, using a confocal microscopy image-based finite element model and simulation of cell stretching, we found that the cell shape is the primary determinant of the strain amplification inside the chondrocyte nucleus in the dedifferentiated state. Additionally, we found that nuclear envelope proteins have lower gene expression in the dedifferentiated state suggesting a weaker nuclear envelope which can further intensify the intranuclear strain amplification. Our results indicate that dedifferentiation and altered nuclear strain could promote gene expression changes at the nuclear envelope, thus promoting further deviation from chondrocyte phenotype. This study highlights the role of cell shape on nuclear mechanics and lays the groundwork to design biophysical strategies for the maintenance and enhancement of the chondrocyte phenotype during expansion with a goal of successful cartilage tissue engineering.SIGNIFICANCEChondrocytes dedifferentiate into a fibroblast-like phenotype in a non-native biophysical environment. Using high resolution microscopy, intranuclear strain analysis, finite element method based computational modeling, and molecular biology techniques, we investigated how mechanical force causes abnormal intranuclear strain distribution in chondrocytes during the dedifferentiation process. Overall, our results suggest that the altered cell geometry aided by an altered or weakened nuclear envelope structure are responsible for abnormal intranuclear strain during chondrocyte dedifferentiation that can further deviate chondrocytes to a more dedifferentiated state.