The extracellular matrix is a key component of tissues, yet it is underrepresented in proteomic datasets. Identification and evaluation of proteins in the extracellular matrix (ECM) has proved challenging due to the insolubility of many ECM proteins in traditional protein extraction buffers. Here we separate the decellularization and ECM extraction steps of several prominent methods for evaluation under real-world conditions. The results are used to optimize a two-fraction ECM extraction method. Approximately one dozen additional parameters are tested, and recommendations for analysis based on overall ECM coverage or specific ECM classes are given. Compared with a standard in-solution digest, the optimized method yielded a fourfold improvement in unique ECM peptide identifications.

All animal cells use the motor cytoplasmic dynein 1 (dynein) to transport diverse cargo toward microtubule minus ends and to organize and position microtubule arrays such as the mitotic spindle. Cargo-specific adaptors engage with dynein to recruit and activate the motor, but the molecular mechanisms remain incompletely understood. Here, we use structural and dynamic nuclear magnetic resonance (NMR) analysis to demonstrate that the C-terminal region of human dynein light intermediate chain 1 (LIC1) is intrinsically disordered and contains two short conserved segments with helical propensity. NMR titration experiments reveal that the first helical segment (helix 1) constitutes the main interaction site for the adaptors Spindly (SPDL1), bicaudal D homolog 2 (BICD2), and Hook homolog 3 (HOOK3). In vitro binding assays show that helix 1, but not helix 2, is essential in both LIC1 and LIC2 for binding to SPDL1, BICD2, HOOK3, RAB-interacting lysosomal protein (RILP), RAB11 family-interacting protein 3 (RAB11FIP3), ninein (NIN), and trafficking kinesin-binding protein 1 (TRAK1). Helix 1 is sufficient to bind RILP, whereas other adaptors require additional segments preceding helix 1 for efficient binding. Point mutations in the C-terminal helix 1 of Caenorhabditis elegans LIC, introduced by genome editing, severely affect development, locomotion, and life span of the animal and disrupt the distribution and transport kinetics of membrane cargo in axons of mechanosensory neurons, identical to what is observed when the entire LIC C-terminal region is deleted. Deletion of the C-terminal helix 2 delays dynein-dependent spindle positioning in the one-cell embryo but overall does not significantly perturb dynein function. We conclude that helix 1 in the intrinsically disordered region of LIC provides a conserved link between dynein and structurally diverse cargo adaptor families that is critical for dynein function in vivo. The large size and complex organization of animal cells make the correct and efficient distribution of intracellular content a challenge. The solution is to use motor proteins, which harness energy from ATP hydrolysis to walk along actin filaments or microtubules, for directional transport of cargo. The multi-subunit motor cytoplasmic dynein 1 (dynein) is responsible for transport directed toward the minus ends of microtubules. An important question is how dynein is recruited to its diverse cargo, which includes organelles such as endosomes and mitochondria, proteins, and mRNA. In this study, we use nuclear magnetic resonance spectroscopy to show that the light intermediate chain (LIC) subunit of human dynein uses a short helix in its disordered C-terminal region to bind structurally distinct adaptor proteins that connect the motor to specific cargo. We then use genome editing in the animal model C. elegans to demonstrate the functional relevance of the C-terminal LIC helix for dynein-dependent cargo transport in neurons. Thus, dynein recruitment to cargo involves a highly conserved interaction between LIC and adaptor proteins.

The human breast is remarkably plastic and remodels with each birth to produce milk optimally suited for the changing demands of the newborn. This dynamic nature of lactation makes it challenging to study under controlled conditions. Given the health benefits of human milk, models of secretory mammary tissue would offer opportunities to study factors that influence this important food source. First, 3D models of the mammary duct/alveoli (D/A) were designed inspired by shapes found in vivo. Photoresins based on mammary decellularized extracellular matrix (dECM) were optimized to match the mechanical properties of native breast tissue. Next, these D/A models were printed with a volumetric printer and seeded with human milk-derived mammary epithelial cells (MECs). MECs formed stable epithelial layers on the printed surfaces and secreted the β-casein and milk fat globules. This model offers exciting avenues to explore hormonal, nutritional, and mechanobiological factors involved in lactation, thereby improving understanding of lactation for the benefit of infants and their mothers.

Tissue-engineered grafts that mimic articular cartilage show promise for treating cartilage injuries. However, engineering cartilage cell-based therapies to match zonal architecture and biochemical composition remains challenging. Decellularized articular cartilage extracellular matrix (dECM) has gained attention for its chondro-inductive properties, yet dECM-based bioinks have limitations in mechanical stability and printability. This study proposes a rapid light-based bioprinting method using a tyrosine-based crosslinking mechanism, which does not require chemical modifications of dECM and thereby preserves its structure and bioactivity. Combining this resin with Filamented Light (FLight) biofabrication enables the creation of cellular, porous, and anisotropic dECM scaffolds composed of aligned microfilaments. Specifically, we focus on the effects of various biopolymer compositions (i.e. hyaluronic acid, collagen I, and dECM) and inner architecture (i.e. bulk light vs FLight) on immune response and cell morphology, and we investigate their influence on nascent ECM production and long-term tissue maturation. Our findings highlight the importance of FLight scaffolds in directing collagen deposition resembling articular cartilage structure and promoting construct maturation, and they emphasize the superiority of biological-rich dECM over single-component materials for engineering articular cartilage, thereby offering new avenues for the development of effective cartilage tissue engineering strategies.

The extracellular matrix is a critical non-cellular component of multi-cellular organisms containing a variety of proteins, glycoproteins, and proteoglycans which has been implicated in a wide variety of essential biological processes, including development, wound healing, and aging. Due to low solubility, many ECM proteins have been underrepresented in previous proteomics datasets. Using an optimized 3-step decellularization and ECM extraction method involving chaotrope extraction and digestion via hydroxylamine hydrochloride, we have generated coverage of the matrisome across 25 organs. We observe that the top 100 most abundant proteins from the ECM fractions of all tissues are generally present in all tissues, indicating that tissue matrices are principally composed of a shared set of ECM proteins. However, these proteins vary up to 4,000-fold between tissues, resulting in highly unique matrix profiles even with the same primary set of proteins. A data reduction approach was used to reveal related networks of expressed ECM proteins across varying tissues, including basement membrane and collagen subtypes.