Cep104 is a component of the centriole distal tip complex that regulates centriole growth and contributes to Drosophila spermiogenesis.

Proper centrosome number and function relies on the accurate assembly of centrioles, barrel-shaped structures that form the core duplicating elements of the organelle. The growth of centrioles is regulated in a cell cycle-dependent manner; while new daughter centrioles elongate during the S/G2/M phase, mature mother centrioles maintain their length throughout the cell cycle. Centriole length is controlled by the synchronized growth of the microtubules that ensheathe the centriole barrel. Although proteins exist that target the growing distal tips of centrioles, such as CP110 and Cep97, these proteins are generally thought to suppress centriolar microtubule growth, suggesting that distal tips may also contain unidentified counteracting factors that facilitate microtubule polymerization. Currently, a mechanistic understanding of how distal tip proteins balance microtubule growth and shrinkage to either promote daughter centriole elongation or maintain centriole length is lacking. Using a proximity-labeling screen in Drosophila cells, we identified Cep104 as a novel component of a group of evolutionarily conserved proteins that we collectively refer to as the distal tip complex (DTC). We found that Cep104 regulates centriole growth and promotes centriole elongation through its microtubule-binding TOG domain. Furthermore, analysis of Cep104 null flies revealed that Cep104 and Cep97 cooperate during spermiogenesis to align spermatids and coordinate individualization. Lastly, we mapped the complete DTC interactome and showed that Cep97 is the central scaffolding unit required to recruit DTC components to the distal tip of centrioles.

Polo-like kinase 4 homodimerization and condensate formation regulate its own protein levels but are not required for centriole assembly.

Polo-like kinase 4 (Plk4) is the master-regulator of centriole assembly, and cell cycle-dependent regulation of its activity maintains proper centrosome number. During most of the cell cycle, Plk4 levels are nearly undetectable due to its ability to autophosphorylate and trigger its own ubiquitin-mediated degradation. However, during mitotic exit, Plk4 forms a single aggregate on the centriole surface to stimulate centriole duplication. Whereas most Polo-like kinase family members are monomeric, Plk4 is unique because it forms homodimers. Notably, Plk4 trans-autophosphorylates a degron near its kinase domain, a critical step in autodestruction. While it is thought that the purpose of homodimerization is to promote trans-autophosphorylation, this has not been tested. Here, we generated separation-of-function Plk4 mutants that fail to dimerize and show that homodimerization creates a binding site for the Plk4 activator, Asterless. Surprisingly, however, Plk4 dimer mutants are catalytically active in cells, promote centriole assembly, and can trans-autophosphorylate through concentration-dependent condensate formation. Moreover, we mapped and then deleted the weak-interacting regions within Plk4 that mediate condensation and conclude that dimerization and condensation are not required for centriole assembly. Our findings suggest that Plk4 dimerization and condensation function simply to down-regulate Plk4 and suppress centriole overduplication.

Predicting Resolvin D1 Pharmacokinetics in Humans with Physiologically-Based Pharmacokinetic Modeling.

Sjögren's syndrome (SS) is an autoimmune disease with no effective treatment options. Resolvin D1 (RvD1) belongs to a class of lipid-based specialized pro-resolving mediators that showed efficacy in preclinical models of SS. We developed a physiologically-based pharmacokinetic (PBPK) model of RvD1 in mice and optimized the model using plasma and salivary gland pharmacokinetic (PK) studies performed in NOD/ShiLtJ mice with SS-like features. The predictive performance of the PBPK model was also evaluated with two external datasets from the literature reporting RvD1 PKs. The PBPK model adequately captured the observed concentrations of RvD1 administered at different doses and in different species. The PKs of RvD1 in virtual humans were predicted using the verified PBPK model at various doses (0.01-10 mg/kg). The first-in-human predictions of RvD1 will be useful for the clinical trial design and translation of RvD1 as an effective treatment strategy for SS.

Sex-dependent Regeneration Patterns in Mouse Submandibular Glands.

Our previous studies indicated that YIGSR-A99 peptides chemically conjugated to fibrin hydrogel (FH) and applied to wounded submandibular gland (SMG) in vivo, formed new organized salivary tissue, whereas wounded SMG treated with FH alone or in the absence of a scaffold showed disorganized collagen formation and poor tissue healing. While these studies indicated that damaged SMG grow and differentiate when treated with FH containing L1 peptide, they were performed only in female mice. However, there is a well-established sexual dimorphism present in mouse SMG (e.g., males develop well-differentiated granular convoluted tubules, but these structures are poorly developed in females) and little is known about how these sex differences influence wound healing events. Therefore, the goal of this study was to conduct comparative analyses of regeneration patterns in male and female mice using L1p-FH in a wounded SMG mouse model. Particularly, we focused on sex-dependent wound healing events such as macrophage polarization, vascularization, tissue organization, and collagen deposition, and how these events affect salivary gland functioning.

Using cell sheets to regenerate mouse submandibular glands.

Temperature-responsive polymer grafted tissue culture dishes release cells as confluent living sheets in response to small changes in temperature, with recovered cell sheets retaining cell-cell communications, functional extracellular matrices and tissue-like behaviors. These features promote tissue regeneration and improve transplantation efficacy in various tissues including cartilage, heart, kidney, liver, endometrium, cornea, middle ear, periodontium, and esophageal living sheet transplants. However, the functional effects of cell sheets for salivary gland regeneration to treat hyposalivation have not yet been studied. Thus, the present study aims to both establish the viability of thermoresponsive cell sheets for use in salivary glands and then explore the delivery option (i.e., single vs. multiple layers) that would result in the most complete tissue growth in terms of cell differentiation and recovered tissue integrity. Results indicate that single cell sheets form polarized structures that maintain cell-cell junctions and secretory granules in vitro while layering of two-single cell sheets forms a glandular-like pattern in vitro. Moreover, double layer cell sheets enhance tissue formation, cell differentiation and saliva secretion in vivo. In contrast, single cell sheets demonstrated only modest gains relative to the robust growth seen with the double layer variety. Together, these data verify the utility of thermoresponsive cell sheets for use in salivary glands and indicates the double layer form to provide the best option in terms of cell differentiation and recovered tissue integrity, thereby offering a potential new therapeutic strategy for treating hyposalivation.

Synergistic effects of laminin-1 peptides, VEGF and FGF9 on salivary gland regeneration.

Hyposalivation is associated with radiation therapy, Sjögren's syndrome and/or aging, and is a significant clinical problem that decreases oral health and overall health in many patients and currently lacks effective treatment. Hence, methods to regenerate salivary glands and restore saliva secretion are urgently needed. To this end, this study describes the modification of fibrin hydrogels with a combination of laminin-1 peptides (YIGSR and A99) and human growth factors (vascular endothelial growth factor and fibroblast growth factor 9) to enhance regeneration in a salivary gland injury mouse model. Our results indicate that these fortified hydrogels enhanced angiogenesis and neurogenesis while promoting formation of acinar structures, thereby leading to enhanced saliva secretion. Such functional recovery indicates salivary gland regeneration and suggests that our technology may be useful in promoting gland regeneration and reversing hyposalivation in a clinical setting. STATEMENT OF SIGNIFICANCE: We engineered Fibrin Hydrogels (FH) to contain multiple regenerative cues including laminin-1 peptides (L1p) and growth factors (GFs). L1p and GF modified FH were used to induce salivary gland regeneration in a wounded mouse model. Treatment with L1p and GF modified FH promoted salivary epithelial tissue regeneration, vascularization, neurogenesis and healing as compared to L1p-FH or FH alone. Results indicate that L1p and GF modified FH can be used for future therapeutic applications.