Changes in endocytosis and trafficking during proliferative quiescence

A significant proportion of cells in the adult human body, including naïve lymphocytes, hepatocytes, stem cells and cancer stem cells, reside in an actively maintained state of proliferative quiescence (G0). Quiescent cells exit the cell cycle under certain conditions and resume proliferation upon appropriate stimuli, which distinguishes them from senescent or terminally differentiated cells. Little is known about how molecular processes, such as endocytosis, are regulated upon cell cycle exit. The aim of this study was to measure endocytic pathways during quiescence and study how their regulation contributes to quiescence maintenance. A quiescence induction protocol was optimised for hTERT-immortalised RPE1 (retina pigmented epithelial) cells to compare endocytosis between quiescent and proliferating RPE1 cells. A SILAC mass spectrometry screen comparing proteome and phosphoproteome between G0 and G1 RPE1 cells revealed changes in total protein levels and phosphorylation of endocytic proteins during G0. Confocal microscopy, Western blotting, flow cytometry and high throughput imaging techniques were used to measure clathrin-mediated (CME) and -independent endocytosis in quiescent and continuously proliferating RPE1 cells. Total levels of core proteins of the clathrin machinery were increased during G0, but uptake of the classical CME cargoes transferrin, EGF and LDL were decreased. CME activity during quiescence is cargo-specific, as could be shown for elevated Lamp1 endocytosis. Endocytosis of clathrin-independent cargoes such as oxidised LDL and Cholera toxin was highly active in quiescent cells, as was uptake of the acropinocytosis cargoes dextran and BSA. Elevated BSA uptake, however, did not promote mTORC1-mediated survival in a nutrient-(amino acid-) deprived environment. Moreover, BSA endocytosis was mediated by AP2. Finally, quiescence survival signalling via integrins was found to be dependent on endocytosis and recycling. Together, this study identified differentially regulated endocytic pathways and suggestd a role for integrin trafficking to maintain proliferative quiescence

Numerical approximation of phase field based shape and topology optimization for fluids

We consider the problem of finding optimal shapes of fluid domains. The fluid obeys the Navier--Stokes equations. Inside a holdall container we use a phase field approach using diffuse interfaces to describe the domain of free flow. We formulate a corresponding optimization problem where flow outside the fluid domain is penalized. The resulting formulation of the shape optimization problem is shown to be well-posed, hence there exists a minimizer, and first order optimality conditions are derived. For the numerical realization we introduce a mass conserving gradient flow and obtain a Cahn--Hilliard type system, which is integrated numerically using the finite element method. An adaptive concept using reliable, residual based error estimation is exploited for the resolution of the spatial mesh. The overall concept is numerically investigated and comparison values are provided

Shape optimization for surface functionals in Navier--Stokes flow using a phase field approach

We consider shape and topology optimization for fluids which are governed by the Navier--Stokes equations. Shapes are modelled with the help of a phase field approach and the solid body is relaxed to be a porous medium. The phase field method uses a Ginzburg--Landau functional in order to approximate a perimeter penalization. We focus on surface functionals and carefully introduce a new modelling variant, show existence of minimizers and derive first order necessary conditions. These conditions are related to classical shape derivatives by identifying the sharp interface limit with the help of formally matched asymptotic expansions. Finally, we present numerical computations based on a Cahn--Hilliard type gradient descent which demonstrate that the method can be used to solve shape optimization problems for fluids with the help of the new approach

Endocytosis in proliferating, quiescent and terminally differentiated cells

Endocytosis mediates nutrient uptake, receptor internalization and the regulation of cell signaling. It is also hijacked by many bacteria, viruses and toxins to mediate their cellular entry. Several endocytic routes exist in parallel, fulfilling different functions. Most studies on endocytosis have used transformed cells in culture. However, as the majority of cells in an adult body have exited the cell cycle, our understanding is biased towards proliferating cells. Here, we review the evidence for the different pathways of endocytosis not only in dividing, but also in quiescent, senescent and terminally differentiated cells. During mitosis, residual endocytosis is dedicated to the internalization of caveolae and specific receptors. In non-dividing cells, clathrin-mediated endocytosis (CME) functions, but the activity of alternative processes, such as caveolae, macropinocytosis and clathrin-independent routes, vary widely depending on cell types and functions. Endocytosis supports the quiescent state by either upregulating cell cycle arrest pathways or downregulating mitogen-induced signaling, thereby inhibiting cell proliferation. Endocytosis in terminally differentiated cells, such as skeletal muscles, adipocytes, kidney podocytes and neurons, supports tissue-specific functions. Finally, uptake is downregulated in senescent cells, making them insensitive to proliferative stimuli by growth factors. Future studies should reveal the molecular basis for the differences in activities between the different cell states

The CTLA-4 immune checkpoint protein regulates PD-L1:PD-1 interaction via transendocytosis of its ligand CD80

CTLA-4 and PD-1 are key immune checkpoint receptors that are targeted in the treatment of cancer. A recently identified physical interaction between the respective ligands, CD80 and PD-L1, has been shown to block PD-L1/PD-1 binding and to prevent PD-L1 inhibitory functions. Since CTLA-4 is known to capture and degrade its ligands via transendocytosis, we investigated the interplay between CD80 transendocytosis and CD80/PD-L1 interaction. We find that transendocytosis of CD80 results in a time-dependent recovery of PD-L1 availability that correlates with CD80 removal. Moreover, CD80 transendocytosis is highly specific in that only CD80 is internalised, while its heterodimeric PD-L1 partner remains on the plasma membrane of the antigen-presenting cell (APC). CTLA-4 interactions with CD80 do not appear to be inhibited by PD-L1, but efficient removal of CD80 requires an intact CTLA-4 cytoplasmic domain, distinguishing this process from more general trogocytosis and simple CTLA-4 binding to CD80/PD-L1 complexes. These data are consistent with CTLA-4 acting as modulator of PD-L1:PD-1 interactions via control of CD80

IL-21 shapes germinal center polarization via light zone B cell selection and cyclin D3 upregulation

Germinal center (GC) dysregulation has been widely reported in the context of autoimmunity. Here, we show that interleukin 21 (IL-21), the archetypal follicular helper T cell (Tfh) cytokine, shapes the scale and polarization of spontaneous chronic autoimmune as well as transient immunization-induced GC. We find that IL-21 receptor deficiency results in smaller GC that are profoundly skewed toward a light zone GC B cell phenotype and that IL-21 plays a key role in selection of light zone GC B cells for entry to the dark zone. Light zone skewing has been previously reported in mice lacking the cell cycle regulator cyclin D3. We demonstrate that IL-21 triggers cyclin D3 upregulation in GC B cells, thereby tuning dark zone inertial cell cycling. Lastly, we identify Foxo1 regulation as a link between IL-21 signaling and GC dark zone formation. These findings reveal new biological roles for IL-21 within GC and have implications for autoimmune settings where IL-21 is overproduced

Development of a biodegradable microstent for minimally invasive treatment of Fallopian tube occlusions

Obstructions of the Fallopian tube represent one of the most common reasons for an unfulfilled desire to have children. Microstent technology opens up new therapeutic possibilities to restore the natural lumen of the Fallopian tube within a single treatment. Within the current work we developed a self-expandable biodegradable microstent for gynecological applications. Based on a novel microstent design, prototypes were manufactured from poly-L-lactide tubing by means of fs-laser cutting. Microstent prototypes were characterized morphologically by means of scanning electron microscopy and biaxial laser scanning. As manufactured, a microstents outside diameter of about 2.3 mm and a strut thickness/width of about 114 µm/103 µm was measured. Mechanical characterization of microstents included bending as well as crimping and release behavior. After crimping to a minimum diameter of 0.8 mm and consecutive release, a microstent recovery to a diameter of 1.8 mm was found. Therefore, proof-of-concept for the self-expandable microstent could be successfully provided. © 2020 by Walter de Gruyter Berlin/Boston 2020

Therapeutic gene editing of T cells to correct CTLA-4 insufficiency

Heterozygous mutations in CTLA-4 result in an inborn error of immunity with an autoimmune and frequently severe clinical phenotype. Autologous T cell gene therapy may offer a cure without the immunological complications of allogeneic hematopoietic stem cell transplantation. Here, we designed a homology-directed repair (HDR) gene editing strategy that inserts the CTLA-4 cDNA into the first intron of the CTLA-4 genomic locus in primary human T cells. This resulted in regulated expression of CTLA-4 in CD4+ T cells, and functional studies demonstrated CD80 and CD86 transendocytosis. Gene editing of T cells isolated from three patients with CTLA-4 insufficiency also restored CTLA-4 protein expression and rescued transendocytosis of CD80 and CD86 in vitro. Last, gene-corrected T cells from CTLA-4-/- mice engrafted and prevented lymphoproliferation in an in vivo murine model of CTLA-4 insufficiency. These results demonstrate the feasibility of a therapeutic approach using T cell gene therapy for CTLA-4 insufficiency

Differences in CD80 and CD86 transendocytosis reveal CD86 as a key target for CTLA-4 immune regulation

CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity