The Rab11 effector Rab11-family interacting protein 2 (Rab11-FIP2) regulates transcytosis through its interactions with Rab11a and myosin Vb. Previous studies implicated Rab11-FIP2 in the establishment of polarity in Madin-Darby canine kidney (MDCK) cells through phosphorylation of Ser-227 by MARK2. Here we examine the dynamic role of Rab11-FIP2 phosphorylation on MDCK cell polarity. Endogenous Rab11-FIP2 phosphorylated on Ser-227 coalesces on vesicular plaques during the reestablishment of polarity after either monolayer wounding or calcium switch. Whereas expression of the nonphosphorylatable Rab11-FIP2(S227A) elicits a loss in lumen formation in MDCK cell cysts grown in Matrigel, the putative pseudophosphorylated Rab11-FIP2(S227E) mutant induces the formation of cysts with multiple lumens. On permeable filters, Rab11-FIP2(S227E)-expressing cells exhibit alterations in the composition of both the adherens and tight junctions. At the adherens junction, p120 catenin and K-cadherin are retained, whereas the majority of the E-cadherin is lost. Although ZO-1 is retained at the tight junction, occludin is lost and the claudin composition is altered. Of interest, the effects of Rab11-FIP2 on cellular polarity did not involve myosin Vb or Rab11a. These results indicate that Ser-227 phosphorylation of Rab11-FIP2 regulates the composition of both adherens and tight junctions and is intimately involved in the regulation of polarity in epithelial cells.

Cells use multiple pathways to internalize and recycle cell surface components. Although Rab11a and Myosin Vb are involved in the recycling of proteins internalized by clathrin-mediated endocytosis, Rab8a has been implicated in nonclathrin-dependent endocytosis and recycling. By yeast two-hybrid assays, we have now demonstrated that Myosin Vb can interact with Rab8a, but not Rab8b. We have confirmed the interaction of Myosin Vb with Rab11a and Rab8a in vivo by using fluorescent resonant energy transfer techniques. Rab8a and Myosin Vb colocalize to a tubular network containing EHD1 and EHD3, which does not contain Rab11a. Myosin Vb tail can cause the accumulation of both Rab11a and Rab8a in collapsed membrane cisternae, whereas dominant-negative Rab11-FIP2(129-512) selectively accumulates Rab11a but not Rab8a. Additionally, dynamic live cell imaging demonstrates distinct pathways for Rab11a and Rab8a vesicle trafficking. These findings indicate that Rab8a and Rab11a define different recycling pathways that both use Myosin Vb.

Multiplexed imaging is a nascent single-cell assay with a complex data structure susceptible to technical variability that disrupts inference. These in situ methods are valuable in understanding cell-cell interactions, but few standardized processing steps or normalization techniques of multiplexed imaging data are available.

Microvillus inclusion disease (MVID), caused by loss-of-function mutations in the motor protein myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid/base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking antidiarrheal drug crofelemer dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. γ-Secretase inhibition with DAPT recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum/glucocorticoid-regulated kinase 2 (SGK2) and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Analysis of spatial multi-omic data from 31 human colorectal specimens enabled phylogeographic mapping of tumor evolution that revealed individualized progression trajectories and accompanying microenvironmental and clonal alterations. Phylogeographic mapping ordered genetic events, classified tumors by their evolutionary dynamics, and placed clonal regions along global pseudotemporal progression trajectories encompassing the chromosomal instability (CIN+) and hypermutated (HM) pathways. Integrated single-cell and spatial transcriptomic data revealed recurring epithelial programs and infiltrating immune states along progression pseudotime. We discovered an immune exclusion signature (IEX), consisting of extracellular matrix regulators DDR1, TGFBI, PAK4, and DPEP1, that charts with CIN+ tumor progression, is associated with reduced cytotoxic cell infiltration, and shows prognostic value in independent cohorts. This spatial multi-omic atlas provides insights into colorectal tumor-microenvironment co-evolution, serving as a resource for stratification and targeted treatments.

The ventricular-subventricular zone (V-SVZ) of the mammalian brain is a site of adult neurogenesis. Within the V-SVZ reside type B neural stem cells (NSCs) and type A neuroblasts. The V-SVZ is also a primary site for very aggressive glioblastoma (GBM). Standard-of-care therapy for GBM consists of safe maximum resection, concurrent temozolomide (TMZ), and X-irradiation (XRT), followed by adjuvant TMZ therapy. The question of how this therapy impacts neurogenesis is not well understood and is of fundamental importance as normal tissue tolerance is a limiting factor. Here, we studied the effects of concurrent TMZ/XRT followed by adjuvant TMZ on type B stem cells and type A neuroblasts of the V-SVZ in C57BL/6 mice. We found that chemoradiation induced an apoptotic response in type A neuroblasts, as marked by cleavage of caspase 3, but not in NSCs, and that A cells within the V-SVZ were repopulated given sufficient recovery time. 53BP1 foci formation and resolution was used to assess the repair of DNA double-strand breaks. Remarkably, the repair was the same in type B and type A cells. While Bax expression was the same for type A or B cells, antiapoptotic Bcl2 and Mcl1 expression was significantly greater in NSCs. Thus, the resistance of type B NSCs to TMZ/XRT appears to be due, in part, to high basal expression of antiapoptotic proteins compared with type A cells. This preclinical research, demonstrating that murine NSCs residing in the V-SVZ are tolerant of standard chemoradiation therapy, supports a dose escalation strategy for treatment of GBM. Stem Cells 2019;37:1629-1639.

Brain-derived neurotrophic factor (BDNF) is a neuronal growth and survival factor that harbors cardioprotective qualities that may attenuate dilated cardiomyopathy. In ~30% of the population, BDNF has a common, nonsynonymous single nucleotide polymorphism rs6265 (Val66Met), which might be correlated with increased risk of cardiovascular events. We previously showed that BDNF correlates with better cardiac function in Duchenne muscular dystrophy (DMD) patients. However, the effect of the Val66Met polymorphism on cardiac function has not been determined. The goal of the current study was to determine the effects of rs6265 on BDNF biomarker suitability and DMD cardiac functions more generally. We assessed cardiovascular and skeletal muscle function in human DMD patients segregated by polymorphic allele. We also compared echocardiographic, electrophysiologic, and cardiomyocyte contractility in C57/BL-6 wild-type mice with rs6265 polymorphism and in mdx/mTR (mDMD) mouse model of DMD. In human DMD patients, plasma BDNF levels had a positive correlation with left ventricular function, opposite to that seen in rs6265 carriers. There was also a substantial decrease in skeletal muscle function in carriers compared to the Val homozygotes. Surprisingly, the opposite was true when cardiac function of DMD carriers and non-carriers were compared. On the other hand, Val66Met wild-type mice had only subtle functional differences at baseline but significantly decreased cardiomyocyte contractility. Our results indicate that the Val66Met polymorphism alters myocyte contractility, conferring worse skeletal muscle function but better cardiac function in DMD patients. Moreover, these results suggest a mechanism for the relative preservation of cardiac tissues compared to skeletal muscle in DMD patients and underscores the complexity of BDNF signaling in response to mechanical workload.

While immune checkpoint blockade (ICB) is the current first-line treatment for metastatic melanoma, it is effective for ~ 52% of patients and has dangerous side effects. The objective here was to identify the feasibility and mechanism of RAS/RAF/PI3K pathway inhibition in melanoma to sensitize tumors to ICB therapy.

The tumor microenvironment plays a key role in the pathogenesis of colorectal tumors and contains various cell types including epithelial, immune, and mesenchymal cells. Characterization of the interactions between these cell types is necessary for revealing the complex nature of tumors. In this study, we used single-cell RNA-seq (scRNA-seq) to compare the tumor microenvironments between a mouse model of sporadic colorectal adenoma (Lrig1CreERT2/+;Apc2lox14/+) and a mouse model of inflammation-driven colorectal cancer induced by azoxymethane and dextran sodium sulfate (AOM/DSS). While both models develop tumors in the distal colon, we found that the two tumor types have distinct microenvironments. AOM/DSS tumors have an increased abundance of two populations of cancer-associated fibroblasts (CAFs) compared with APC tumors, and we revealed their divergent spatial association with tumor cells using multiplex immunofluorescence (MxIF) imaging. We also identified a unique squamous cell population in AOM/DSS tumors, whose origins were distinct from anal squamous epithelial cells. These cells were in higher proportions upon administration of a chemotherapy regimen of 5-Fluorouracil/Irinotecan. We used computational inference algorithms to predict cell-cell communication mediated by ligand-receptor interactions and downstream pathway activation, and identified potential mechanistic connections between CAFs and tumor cells, as well as CAFs and squamous epithelial cells. This study provides important preclinical insight into the microenvironment of two distinct models of colorectal tumors and reveals unique roles for CAFs and squamous epithelial cells in the AOM/DSS model of inflammation-driven cancer.

Crohn's disease (CD) is a complex chronic inflammatory disorder that may affect any part of gastrointestinal tract with extra-intestinal manifestations and associated immune dysregulation. To characterize heterogeneity in CD, we profiled single-cell transcriptomics of 170 samples from 65 CD patients and 18 non-inflammatory bowel disease (IBD) controls in both the terminal ileum (TI) and ascending colon (AC). Analysis of 202,359 cells identified a novel epithelial cell type in both TI and AC, featuring high expression of LCN2, NOS2, and DUOX2, and thus is named LND. LND cells, confirmed by high-resolution in-situ RNA imaging, were rarely found in non-IBD controls, but expanded significantly in active CD. Compared to other epithelial cells, genes defining LND cells were enriched in antimicrobial response and immunoregulation. Moreover, multiplexed protein imaging demonstrated that LND cell abundance was associated with immune infiltration. Cross-talk between LND and immune cells was explored by ligand-receptor interactions and further evidenced by their spatial colocalization. LND cells showed significant enrichment of expression specificity of IBD/CD susceptibility genes, revealing its role in immunopathogenesis of CD. Investigating lineage relationships of epithelial cells detected two LND cell subpopulations with different origins and developmental potential, early and late LND. The ratio of the late to early LND cells was related to anti-TNF response. These findings emphasize the pathogenic role of the specialized LND cell type in both Crohn's ileitis and Crohn's colitis and identify novel biomarkers associated with disease activity and treatment response.

Chemotherapeutics and other pharmaceuticals are common sources of cellular stress. Darinaparsin (ZIO-101) is a novel organic arsenical under evaluation as a cancer chemotherapeutic, but the drug's precise mechanism of action is unclear. Stress granule formation is an important cellular stress response, but the mechanisms of formation, maintenance, and dispersal of RNA-containing granules are not fully understood. During stress, small, diffuse granules initially form throughout the cytoplasm. These granules then coalesce near the nucleus into larger granules that disperse once the cellular stress is removed. Complete stress granule formation is dependent upon microtubules. Human cervical cancer (HeLa) cells, pre-treated with nocodazole for microtubule depolymerization, formed only small, diffuse stress granules upon sodium arsenite treatment. Darinaparsin, as a single agent, also induced the formation of small, diffuse stress granules, an effect similar to that of the combination of nocodazole with sodium arsenite. Darinaparsin inhibited the polymerization of microtubules both in vivo and in vitro. Interestingly, upon removal of darinaparsin, the small, diffuse stress granules completed formation with coalescence in the perinuclear region prior to disassembly. These results indicate that RNA stress granules must complete formation prior to disassembly, and completion of stress granule formation is dependent upon microtubules. Finally, treatment of cells with darinaparsin led to a reduction in Sonic hedgehog (Shh) stimulated activation of Gli1 and a loss of primary cilia. Therefore, darinaparsin represents a unique multivalent chemotherapeutic acting on stress induction, microtubule polymerization, and Shh signaling.

Rab proteins influence vesicle trafficking pathways through the assembly of regulatory protein complexes. Previous investigations have documented that Rab11a and Rab8a can interact with the tail region of myosin Vb and regulate distinct trafficking pathways. We have now determined that a related Rab protein, Rab10, can interact with myosin Va, myosin Vb, and myosin Vc. Rab10 localized to a system of tubules and vesicles that have partially overlapping localization with Rab8a. Both Rab8a and Rab10 were mislocalized by the expression of dominant-negative myosin V tails. Interaction with Rab10 was dependent on the presence of the alternatively spliced exon D in myosin Va and myosin Vb and the homologous region in myosin Vc. Yeast two-hybrid assays and fluorescence resonance energy transfer studies confirmed that Rab10 binding to myosin V tails in vivo required the alternatively spliced exon D. In contrast to our previous work, we found that Rab11a can interact with both myosin Va and myosin Vb tails independent of their splice isoform. These results indicate that Rab GTPases regulate diverse endocytic trafficking pathways through recruitment of multiple myosin V isoforms.

No abstract available

Microbial influences on host cells depend upon the identities of the microbes, their spatial localization, and the responses they invoke on specific host cell populations. Multimodal analyses of both microbes and host cells in a spatially resolved fashion would enable studies into these complex interactions in native tissue environments, potentially in clinical specimens. While techniques to preserve each of the microbial and host cell compartments have been used to examine tissues and microbes separately, we endeavored to develop approaches to simultaneously analyze both compartments. Herein, we established an original method for mucus preservation using Poloxamer 407 (also known as Pluronic F-127), a thermoreversible polymer with mucus-adhesive characteristics. We demonstrate that this approach can preserve spatially-defined compartments of the mucus bi-layer in the colon and the bacterial communities within, compared with their marked absence when tissues were processed with traditional formalin-fixed paraffin-embedded (FFPE) pipelines. Additionally, antigens for antibody staining of host cells were preserved and signal intensity for 16S rRNA fluorescence in situ hybridization (FISH) was enhanced in poloxamer-fixed samples. This in turn enabled us to integrate multimodal analysis using a modified multiplex immunofluorescence (MxIF) protocol. Importantly, we have formulated Poloxamer 407 to polymerize and cross-link at room temperature for use in clinical workflows. These results suggest that the fixative formulation of Poloxamer 407 can be integrated into biospecimen collection pipelines for simultaneous analysis of microbes and host cells.

Increasingly, highly multiplexed tissue imaging methods are used to profile protein expression at the single-cell level. However, a critical limitation is the lack of robust cell segmentation tools for tissue sections. We present Multiplexed Image Resegmentation of Internal Aberrant Membranes (MIRIAM) that combines (a) a pipeline for cell segmentation and quantification that incorporates machine learning-based pixel classification to define cellular compartments, (b) a novel method for extending incomplete cell membranes, and (c) a deep learning-based cell shape descriptor. Using human colonic adenomas as an example, we show that MIRIAM is superior to widely utilized segmentation methods and provides a pipeline that is broadly applicable to different imaging platforms and tissue types.

Multiplexed immunofluorescence (mIF) is an emerging assay for multichannel protein imaging that can decipher cell-level spatial features in tissues. However, existing automated cell phenotyping methods, such as clustering, face challenges in achieving consistency across experiments and often require subjective evaluation. As a result, mIF analyses often revert to marker gating based on manual thresholding of raw imaging data.

Inactivating mutations in MYO5B cause severe neonatal diarrhea in Microvillus Inclusion Disease. Loss of active MYO5B causes the formation of pathognomonic inclusions and aberrations in brush border enzymes.

The Rab GTPases are the largest family of proteins regulating membrane traffic. Rab proteins form a nidus for the assembly of multiprotein complexes on distinct vesicle membranes to regulate particular membrane trafficking pathways. Recent investigations have demonstrated that Myosin Vb (Myo5B) is an effector for Rab8a, Rab10, and Rab11a, all of which are implicated in regulating different pathways for recycling of proteins to the plasma membrane. It remains unclear how specific interactions of Myo5B with individual Rab proteins can lead to specificity in the regulation of alternate trafficking pathways. We examined the relative contributions of Rab/Myo5B interactions with specific pathways using Myo5B mutants lacking binding to either Rab11a or Rab8a. Myo5B Q1300L and Y1307C mutations abolished Rab8a association, whereas Myo5B Y1714E and Q1748R mutations uncoupled association with Rab11a. Expression of Myo5B tails containing these mutants demonstrated that Rab11a, but not Rab8a, was required for recycling of transferrin in nonpolarized cells. In contrast, in polarized epithelial cyst cultures, Myo5B was required for apical membrane trafficking and de novo lumen formation, dependent on association with both Rab8a and Rab11a. These data demonstrate that different combinations of Rab GTPase association with Myo5B control distinct membrane trafficking pathways.

A tripartite association of Rab11a with both Rab11-FIP2 and MYO5B regulates recycling endosome trafficking. We sought to define the intermolecular interactions required between Rab11-FIP2 and MYO5B. Using a random mutagenesis strategy, we identified point mutations at S229P or G233E in Rab11-FIP2 that caused loss of interaction with MYO5B in yeast two-hybrid assays as well as loss of interaction of Rab11-FIP2(129-356) with MYO5B tail when expressed in HeLa cells. Single mutations or the double S229P/G233E mutation failed to alter the association of full-length Rab11-FIP2 with MYO5B tail in HeLa cells. While EGFP-Rab11-FIP2 wild type colocalized with endogenous MYO5B staining in MDCK cells, EGFP-Rab11-FIP2(S229P/G233E) showed a significant decrease in localization with endogenous MYO5B. Analysis of Rab11a-containing vesicle movement in live HeLa cells demonstrated that when the MYO5B/Rab11-FIP2 association is perturbed by mutation or by Rab11-FIP2 knockdown, vesicle movement is increased in both speed and track length, consistent with an impairment of MYO5B tethering at the cytoskeleton. These results support a critical role for the interaction of MYO5B with Rab11-FIP2 in stabilizing the functional complex with Rab11a, which regulates dynamic movements of membrane recycling vesicles.

Modern single-cell technologies allow multiplexed sampling of cellular states within a tissue. However, computational tools that can infer developmental cell-state transitions reproducibly from such single-cell data are lacking. Here, we introduce p-Creode, an unsupervised algorithm that produces multi-branching graphs from single-cell data, compares graphs with differing topologies, and infers a statistically robust hierarchy of cell-state transitions that define developmental trajectories. We have applied p-Creode to mass cytometry, multiplex immunofluorescence, and single-cell RNA-seq data. As a test case, we validate cell-state-transition trajectories predicted by p-Creode for intestinal tuft cells, a rare, chemosensory cell type. We clarify that tuft cells are specified outside of the Atoh1-dependent secretory lineage in the small intestine. However, p-Creode also predicts, and we confirm, that tuft cells arise from an alternative, Atoh1-driven developmental program in the colon. These studies introduce p-Creode as a reliable method for analyzing large datasets that depict branching transition trajectories.