High-Throughput Mechanobiology Screening Platform Using Micro- and Nanotopography.

We herein demonstrate the first 96-well plate platform to screen effects of micro- and nanotopographies on cell growth and proliferation. Existing high-throughput platforms test a limited number of factors and are not fully compatible with multiple types of testing and assays. This platform is compatible with high-throughput liquid handling, high-resolution imaging, and all multiwell plate-based instrumentation. We use the platform to screen for topographies and drug-topography combinations that have short- and long-term effects on T cell activation and proliferation. We coated nanofabricated "trench-grid" surfaces with anti-CD3 and anti-CD28 antibodies to activate T cells and assayed for interleukin 2 (IL-2) cytokine production. IL-2 secretion was enhanced at 200 nm trench width and >2.3 µm grating pitch; however, the secretion was suppressed at 100 nm width and <0.5 µm pitch. The enhancement on 200 nm grid trench was further amplified with the addition of blebbistatin to reduce contractility. The 200 nm grid pattern was found to triple the number of T cells in long-term expansion, a result with direct clinical applicability in adoptive immunotherapy.

Micropatterning of TCR and LFA-1 ligands reveals complementary effects on cytoskeleton mechanics in T cells.

The formation of the immunological synapse between a T cell and the antigen-presenting cell (APC) is critically dependent on actin dynamics, downstream of T cell receptor (TCR) and integrin (LFA-1) signalling. There is also accumulating evidence that mechanical forces, generated by actin polymerization and/or myosin contractility regulate T cell signalling. Because both receptor pathways are intertwined, their contributions towards the cytoskeletal organization remain elusive. Here, we identify the specific roles of TCR and LFA-1 by using a combination of micropatterning to spatially separate signalling systems and nanopillar arrays for high-precision analysis of cellular forces. We identify that Arp2/3 acts downstream of TCRs to nucleate dense actin foci but propagation of the network requires LFA-1 and the formin FHOD1. LFA-1 adhesion enhances actomyosin forces, which in turn modulate actin assembly downstream of the TCR. Together our data shows a mechanically cooperative system through which ligands presented by an APC modulate T cell activation.

T cell activation is determined by the number of presented antigens.

Antigen recognition is a key event during T cell activation. Here, we introduce nanopatterned antigen arrays that mimic the antigen presenting cell surface during T cell activation. The assessment of activation related events revealed the requirement of a minimal density of 90-140 stimulating major histocompatibility complex class II proteins (pMHC) molecules per µm(2). We demonstrate that these substrates induce T cell responses in a pMHC dose-dependent manner and that the number of presented pMHCs dominates over local pMHC density.

MLN64 transport to the late endosome is regulated by binding to 14-3-3 via a non-canonical binding site.

MLN64 is an integral membrane protein localized to the late endosome and plasma membrane that is thought to function as a mediator of cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria. The protein consists of two distinct domains: an N-terminal membrane-spanning domain that shares homology with the MENTHO protein and a C-terminal steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain that binds cholesterol. To further characterize the MLN64 protein, full-length and truncated proteins were overexpressed in cells and the effects on MLN64 trafficking and endosomal morphology were observed. To gain insight into MLN64 function, affinity chromatography and mass spectrometric techniques were used to identify potential MLN64 interacting partners. Of the 15 candidate proteins identified, 14-3-3 was chosen for further characterization. We show that MLN64 interacts with 14-3-3 in vitro as well as in vivo and that the strength of the interaction is dependent on the 14-3-3 isoform. Furthermore, blocking the interaction through the use of a 14-3-3 antagonist or MLN64 mutagenesis delays the trafficking of MLN64 to the late endosome and also results in the dispersal of endocytic vesicles to the cell periphery. Taken together, these studies have determined that MLN64 is a novel 14-3-3 binding protein and indicate that 14-3-3 plays a role in the endosomal trafficking of MLN64. Furthermore, these studies suggest that 14-3-3 may be the link by which MLN64 exerts its effects on the actin-mediated endosome dynamics.

Inactivation of NPC1L1 causes multiple lipid transport defects and protects against diet-induced hypercholesterolemia.

NPC1L1, a recently identified relative of Niemann-Pick C1, was characterized to determine its subcellular location and potential function(s). NPC1L1 was highly expressed in HepG2 cells and localized in a subcellular vesicular compartment rich in the small GTPase Rab5. mRNA expression profiling revealed significant differences between mouse and man with highest expression found in human liver and significant expression in the small intestine. In contrast, liver expression in mouse was extremely low with mouse small intestine exhibiting the highest NPC1L1 expression. A mouse knock-out model of NPC1L1 was generated and revealed that mice lacking a functional NPC1L1 have multiple lipid transport defects. Surprisingly, lack of NPC1L1 exerts a protective effect against diet-induced hyperlipidemia. Further characterization of cell lines generated from wild-type and knock-out mice revealed that in contrast to wild-type cells, NPC1L1 cells exhibit aberrant plasma membrane uptake and subsequent transport of various lipids, including cholesterol and sphingolipids. Furthermore, lack of NPC1L1 activity causes a deregulation of caveolin transport and localization, suggesting that the observed lipid transport defects may be the indirect result of an inability of NPC1L1 null cells to properly target and/or regulate caveolin expression.

Reduced renin expression and altered gene transcript profiles in multicystic dysplastic kidneys.

PURPOSE: Some patients with multicystic dysplasia experience hypertension in infancy which can be clinically improved with simple nephrectomy. Little is known of the role of renin in multicystic dysplasia and therefore, we studied renin expression in nonhypertensive multicystic dysplasia and compared it with hypertensive multicystic dysplasia. Additionally, global gene transcript profiles were studied in nonhypertensive multicystic dysplasia. MATERIALS AND METHODS: Tissue from 12 patients with multicystic dysplasia without hypertension and 2 patients with hypertensive multicystic dysplasia were compared to normal metanephric mid-term and neonatal kidneys. Renin expression was studied by immunohistochemistry. CD-68, a marker for macrophages, was applied to slides previously stained with renin. GeneChip (Affymetrix, Southern Oaks, California) microarray analysis was performed using total RNA from 2 nonhypertensive multicystic dysplastic kidneys and normal newborn kidney as control. RESULTS: Strong renin staining was present in arteries and arterioles in normal early metanephric kidneys and concentrated in juxtaglomerular cells in neonatal kidneys. Renin was variably decreased in arterioles in multicystic dysplasia and was ectopically expressed in interstitial cells. Renin positive interstitial cells co-expressed CD-68 antigen identifying these cells as macrophages. Ectopic renin production was more pronounced in hypertensive multicystic dysplasia. GeneChip hybridization showed a 60 to 200-fold decrease in expression of renin transcript in multicystic dysplasia relative to normal kidney, concordant with immunohistochemical results. Altered expression of several other candidate transcripts was also identified. CONCLUSIONS: Dramatic renin decrease in multicystic dysplasia suggests that it may participate in the development of dysplastic vessels. Pronounced ectopic renin expression by macrophages in multicystic dysplasia associated with hypertension may be linked to hypertension in multicystic dysplasia. Several other candidate genes whose altered expression may be associated with the pathophysiology of multicystic dysplasia provide intriguing molecular targets for future study.