Piezo1-Regulated Mechanotransduction Controls Flow-Activated Lymphatic Expansion.

BACKGROUND: Mutations in PIEZO1 (Piezo type mechanosensitive ion channel component 1) cause human lymphatic malformations. We have previously uncovered an ORAI1 (ORAI calcium release-activated calcium modulator 1)-mediated mechanotransduction pathway that triggers lymphatic sprouting through Notch downregulation in response to fluid flow. However, the identity of its upstream mechanosensor remains unknown. This study aimed to identify and characterize the molecular sensor that translates the flow-mediated external signal to the Orai1-regulated lymphatic expansion. METHODS: Various mutant mouse models, cellular, biochemical, and molecular biology tools, and a mouse tail lymphedema model were employed to elucidate the role of Piezo1 in flow-induced lymphatic growth and regeneration. RESULTS: Piezo1 was found to be abundantly expressed in lymphatic endothelial cells. Piezo1 knockdown in cultured lymphatic endothelial cells inhibited the laminar flow-induced calcium influx and abrogated the flow-mediated regulation of the Orai1 downstream genes, such as KLF2 (Krüppel-like factor 2), DTX1 (Deltex E3 ubiquitin ligase 1), DTX3L (Deltex E3 ubiquitin ligase 3L,) and NOTCH1 (Notch receptor 1), which are involved in lymphatic sprouting. Conversely, stimulation of Piezo1 activated the Orai1-regulated mechanotransduction in the absence of fluid flow. Piezo1-mediated mechanotransduction was significantly blocked by Orai1 inhibition, establishing the epistatic relationship between Piezo1 and Orai1. Lymphatic-specific conditional Piezo1 knockout largely phenocopied sprouting defects shown in Orai1- or Klf2- knockout lymphatics during embryo development. Postnatal deletion of Piezo1 induced lymphatic regression in adults. Ectopic Dtx3L expression rescued the lymphatic defects caused by Piezo1 knockout, affirming that the Piezo1 promotes lymphatic sprouting through Notch downregulation. Consistently, transgenic Piezo1 expression or pharmacological Piezo1 activation enhanced lymphatic sprouting. Finally, we assessed a potential therapeutic value of Piezo1 activation in lymphatic regeneration and found that a Piezo1 agonist, Yoda1, effectively suppressed postsurgical lymphedema development. CONCLUSIONS: Piezo1 is an upstream mechanosensor for the lymphatic mechanotransduction pathway and regulates lymphatic growth in response to external physical stimuli. Piezo1 activation presents a novel therapeutic opportunity for preventing postsurgical lymphedema. The Piezo1-regulated lymphangiogenesis mechanism offers a molecular basis for Piezo1-associated lymphatic malformation in humans.

Generation of FLAG-tagged Arx knock-in mouse model.

The Aristaless-related homeobox (ARX) is a paired-like homeodomain transcription factor playing important roles in brain development. Patients with mutations in ARX have a spectrum of neurodevelopmental disorders such as epilepsy, intellectual disability, and autism spectrum disorder, with or without structural abnormalities of the brain such as lissencephaly (smooth brain), microcephaly (small brain), and/or agenesis of the corpus callosum. Mouse models have provided important clues on the pathophysiologic roles of ARX in these disorders. However, successfully isolating specific in vivo complexes of ARX, with DNA and proteins, has remained as a challenge. To facilitate in vivo detection of ARX complexes, we generated a mouse line containing one epitope of FLAG-tag (1 × FLAG) targeted at the translational start site of the endogenous Arx gene using CRSPR/Cas9 strategy. Homozygous Flag-Arx mice are viable and fertile without gross abnormality, suggesting that the FLAG-tag does not perturb the normal function of ARX. Using a FLAG antibody, we successfully detected ARX with immunofluorescent staining and pulled down ARX in embryonic brain tissues. This Flag-Arx mouse line will be a useful tool to isolate ARX complexes from mouse tissues for many applications.

Ascorbate peroxidase proximity labeling coupled with biochemical fractionation identifies promoters of endoplasmic reticulum-mitochondrial contacts.

To maintain cellular homeostasis, subcellular organelles communicate with each other and form physical and functional networks through membrane contact sites coupled by protein tethers. In particular, endoplasmic reticulum (ER)-mitochondrial contacts (EMC) regulate diverse cellular activities such as metabolite exchange (Ca2+ and lipids), intracellular signaling, apoptosis, and autophagy. The significance of EMCs has been highlighted by reports indicating that EMC dysregulation is linked to neurodegenerative diseases. Therefore, obtaining a better understanding of the physical and functional components of EMCs should provide new insights into the pathogenesis of several neurodegenerative diseases. Here, we applied engineered ascorbate peroxidase (APEX) to map the proteome at EMCs in live HEK293 cells. APEX was targeted to the outer mitochondrial membrane, and proximity-labeled proteins were analyzed by stable isotope labeling with amino acids in culture (SILAC)-LC/MS-MS. We further refined the specificity of the proteins identified by combining biochemical subcellular fractionation to the protein isolation method. We identified 405 proteins with a 2.0-fold cutoff ratio (log base 2) in SILAC quantification from replicate experiments. We performed validation screening with a Split-Rluc8 complementation assay that identified reticulon 1A (RTN1A), an ER-shaping protein localized to EMCs as an EMC promoter. Proximity mapping augmented with biochemical fractionation and additional validation methods reported here could be useful to discover other components of EMCs, identify mitochondrial contacts with other organelles, and further unravel their communication.

Genetic Variants That Predispose to DNA Double-Strand Breaks in Lymphocytes From a Subset of Patients With Familial Colorectal Carcinomas.

BACKGROUND & AIMS: DNA structural lesions are prevalent in sporadic colorectal cancer. Therefore, we proposed that gene variants that predispose to DNA double-strand breaks (DSBs) would be found in patients with familial colorectal carcinomas of an undefined genetic basis (UFCRC). METHODS: We collected primary T cells from 25 patients with UFCRC and matched patients without colorectal cancer (controls) and assayed for DSBs. We performed exome sequence analyses of germline DNA from 20 patients with UFCRC and 5 undiagnosed patients with polyposis. The prevalence of identified variants in genes linked to DNA integrity was compared with that of individuals without a family history of cancer. The effects of representative variants found to be associated with UFCRC was confirmed in functional assays with HCT116 cells. RESULTS: Primary T cells from most patients with UFCRC had increased levels of the DSB marker γ(phosphorylated)histone2AX (γH2AX) after treatment with DNA damaging agents, compared with T cells from controls (P < .001). Exome sequence analysis identified a mean 1.4 rare variants per patient that were predicted to disrupt functions of genes relevant to DSBs. Controls (from public databases) had a much lower frequency of variants in the same genes (P < .001). Knockdown of representative variant genes in HCT116 CRC cells increased γH2AX. A detailed analysis of immortalized patient-derived B cells that contained variants in the Werner syndrome, RecQ helicase-like gene (WRN, encoding T705I), and excision repair cross-complementation group 6 (ERCC6, encoding N180Y) showed reduced levels of these proteins and increased DSBs, compared with B cells from controls. This phenotype was rescued by exogenous expression of WRN or ERCC6. Direct analysis of the recombinant variant proteins confirmed defective enzymatic activities. CONCLUSIONS: These results provide evidence that defects in suppression of DSBs underlie some cases of UFCRC; these can be identified by assays of circulating lymphocytes. We specifically associated UFCRC with variants in WRN and ERCC6 that reduce the capacity for repair of DNA DSBs. These observations could lead to a simple screening strategy for UFCRC, and provide insight into the pathogenic mechanisms of colorectal carcinogenesis.

Hereditary spastic paraplegia-linked REEP1 modulates endoplasmic reticulum/mitochondria contacts.

OBJECTIVE: Mutations in receptor expression enhancing protein 1 (REEP1) are associated with hereditary spastic paraplegias (HSPs). Although axonal degeneration is thought to be a predominant feature in HSP, the role of REEP1 mutations in degeneration is largely unknown. Previous studies have implicated a role for REEP1 in the endoplasmic reticulum (ER), whereas others localized REEP1 with mitochondria. We sought to resolve the cellular localization of REEP1 and further elucidate the pathobiology underlying REEP1 mutations in patients. METHODS: A combination of cellular imaging and biochemical approaches was used to refine the cellular localization of REEP1. Next, Reep1 mutations associated with HSP were functionally tested in neuritic growth and degeneration assays using mouse cortical culture. Finally, a novel assay was developed and used with wild-type and mutant Reep1s to measure the interactions between the ER and mitochondria. RESULTS: We found that REEP1 is present at the ER-mitochondria interface, and it contains subdomains for mitochondrial as well as ER localization. Knockdown of Reep1 and expression of pathological Reep1 mutations resulted in neuritic growth defects and degeneration. Finally, using our novel split-RLuc8 assay, we show that REEP1 facilitates ER-mitochondria interactions, a function diminished by disease-associated mutations. INTERPRETATION: Our data potentially reconcile the current conflicting reports regarding REEP1 being either an ER or a mitochondrial protein. Furthermore, our results connect, for the first time, the disrupted ER-mitochondria interactions to a failure in maintaining health of long axons in HSPs. Finally, the split-RLuc8 assay offers a new tool to identify potential drugs for multiple neurodegenerative diseases with ER-mitochondria interaction defects.

ATP-dependent chromatin remodeling by Cockayne syndrome protein B and NAP1-like histone chaperones is required for efficient transcription-coupled DNA repair.

The Cockayne syndrome complementation group B (CSB) protein is essential for transcription-coupled DNA repair, and mutations in CSB are associated with Cockayne syndrome--a devastating disease with complex clinical features, including the appearance of premature aging, sun sensitivity, and numerous neurological and developmental defects. CSB belongs to the SWI2/SNF2 ATP-dependent chromatin remodeler family, but the extent to which CSB remodels chromatin and whether this activity is utilized in DNA repair is unknown. Here, we show that CSB repositions nucleosomes in an ATP-dependent manner in vitro and that this activity is greatly enhanced by the NAP1-like histone chaperones, which we identify as new CSB-binding partners. By mapping functional domains and analyzing CSB derivatives, we demonstrate that chromatin remodeling by the combined activities of CSB and the NAP1-like chaperones is required for efficient transcription-coupled DNA repair. Moreover, we show that chromatin remodeling and repair protein recruitment mediated by CSB are separable activities. The collaboration that we observed between CSB and the NAP1-like histone chaperones adds a new dimension to our understanding of the ways in which ATP-dependent chromatin remodelers and histone chaperones can regulate chromatin structure. Taken together, the results of this study offer new insights into the functions of chromatin remodeling by CSB in transcription-coupled DNA repair as well as the underlying mechanisms of Cockayne syndrome.

Arx together with FoxA2, regulates Shh floor plate expression.

Mutations in the Aristaless related homeodomain transcription factor (ARX) are associated with a diverse set of X-linked mental retardation and epilepsy syndromes in humans. Although most studies have been focused on its function in the forebrain, ARX is also expressed in other regions of the developing nervous system including the floor plate (FP) of the spinal cord where its function is incompletely understood. To investigate the role of Arx in the FP, we performed gain-of-function studies in the chick using in ovo electroporation, and loss-of-function studies in Arx-deficient mice. We have found that Arx, in conjunction with FoxA2, directly induces Sonic hedgehog (Shh) expression through binding to a Shh floor plate enhancer (SFPE2). We also observed that FoxA2 induces Arx through its transcriptional activation domain whereas Nkx2.2, induced by Shh, abolishes this induction. Our data support a feedback loop model for Arx function; through interactions with FoxA2, Arx positively regulates Shh expression in the FP, and Shh signaling in turn activates Nkx2.2, which suppresses Arx expression. Furthermore, our data are evidence that Arx plays a role as a context dependent transcriptional activator, rather than a primary inducer of Shh expression, potentially explaining how mutations in ARX are associated with diverse, and often subtle, defects.

Genetic and functional interactions between Mus81-Mms4 and Rad27.

The two endonucleases, Rad27 (yeast Fen1) and Dna2, jointly participate in the processing of Okazaki fragments in yeasts. Mus81-Mms4 is a structure-specific endonuclease that can resolve stalled replication forks as well as toxic recombination intermediates. In this study, we show that Mus81-Mms4 can suppress dna2 mutational defects by virtue of its functional and physical interaction with Rad27. Mus81-Mms4 stimulated Rad27 activity significantly, accounting for its ability to restore the growth defects caused by the dna2 mutation. Interestingly, Rad27 stimulated the rate of Mus81-Mms4 catalyzed cleavage of various substrates, including regressed replication fork substrates. The ability of Rad27 to stimulate Mus81-Mms4 did not depend on the catalytic activity of Rad27, but required the C-terminal 64 amino acid fragment of Rad27. This indicates that the stimulation was mediated by a specific protein-protein interaction between the two proteins. Our in vitro data indicate that Mus81-Mms4 and Rad27 act together during DNA replication and resolve various structures that can impede normal DNA replication. This conclusion was further strengthened by the fact that rad27 mus81 or rad27 mms4 double mutants were synergistically lethal. We discuss the significance of the interactions between Rad27, Dna2 and Mus81-Mms4 in context of DNA replication.

ß2-adrenergic receptor regulates ER-mitochondria contacts.

Interactions between the endoplasmic reticulum (ER) and mitochondria (Mito) are crucial for many cellular functions, and their interaction levels change dynamically depending on the cellular environment. Little is known about how the interactions between these organelles are regulated within the cell. Here we screened a compound library to identify chemical modulators for ER-Mito contacts in HEK293T cells. Multiple agonists of G-protein coupled receptors (GPCRs), beta-adrenergic receptors (ß-ARs) in particular, scored in this screen. Analyses in multiple orthogonal assays validated that ß2-AR activation promotes physical and functional interactions between the two organelles. Furthermore, we have elucidated potential downstream effectors mediating ß2-AR-induced ER-Mito contacts. Together our study identifies ß2-AR signaling as an important regulatory pathway for ER-Mito coupling and highlights the role of these contacts in responding to physiological demands or stresses.

The Lymphatic Cell Environment Promotes Kaposi Sarcoma Development by Prox1-Enhanced Productive Lytic Replication of Kaposi Sarcoma Herpes Virus.

Kaposi sarcoma is the most common cancer in human immunodeficiency virus-positive individuals and is caused by Kaposi sarcoma-associated herpesvirus (KSHV). It is believed that a small number of latently infected Kaposi sarcoma tumor cells undergo spontaneous lytic reactivation to produce viral progeny for infection of new cells. Here, we use matched donor-derived human dermal blood and lymphatic endothelial cells (BEC and LEC, respectively) to show that KSHV-infected BECs progressively lose viral genome as they proliferate. In sharp contrast, KSHV-infected LECs predominantly entered lytic replication, underwent cell lysis, and released new virus. Continuous lytic cell lysis and de novo infection allowed LEC culture to remain infected for a prolonged time. Because of the strong propensity of LECs toward lytic replication, LECs maintained virus as a population, despite the death of individual host cells from lytic lysis. The master regulator of lymphatic development, Prox1, bound the promoter of the RTA gene to upregulate its expression and physically interacted with RTA protein to coregulate lytic genes. Thus, LECs may serve as a proficient viral reservoir that provides viral progeny for continuous de novo infection of tumor origin cells, and potentially BECs and mesenchymal stem cells, which give rise to Kaposi sarcoma tumors. Our study reveals drastically different host cell behaviors between BEC and LEC and defines the underlying mechanisms of the lymphatic cell environment supporting persistent infection in Kaposi sarcoma tumors. SIGNIFICANCE: This study defines the mechanism by which Kaposi's sarcoma could be maintained by virus constantly produced by lymphatic cells in HIV-positive individuals.