Adaptor protein (AP) complexes mediate key sorting decisions in the cell through selective incorporation of transmembrane proteins into vesicles. Little is known of the roles of AP-4, despite its loss of function leading to a severe early onset neurological disorder, AP-4 deficiency syndrome. Here we demonstrate an AP-4 epsilon subunit knockout mouse model that recapitulates characteristic neuroanatomical phenotypes of AP-4 deficiency patients. We show that ATG9A, critical for autophagosome biogenesis, is an AP-4 cargo, which is retained within the trans-Golgi network (TGN) in vivo and in culture when AP-4 function is lost. TGN retention results in depletion of axonal ATG9A, leading to defective autophagosome generation and aberrant expansions of the distal axon. The reduction in the capacity to generate axonal autophagosomes leads to defective axonal extension and de novo generation of distal axonal swellings containing accumulated ER, underlying the impaired axonal integrity in AP-4 deficiency syndrome.
Summary Altered excitatory/inhibitory (E/I) balance is implicated in neuropsychiatric and neurodevelopmental disorders, but the underlying genetic etiology remains poorly understood. Copy number variations in CYFIP1 are associated with autism, schizophrenia, and intellectual disability, but its role in regulating synaptic inhibition or E/I balance remains unclear. We show that CYFIP1, and the paralog CYFIP2, are enriched at inhibitory postsynaptic sites. While CYFIP1 or CYFIP2 upregulation increases excitatory synapse number and the frequency of miniature excitatory postsynaptic currents (mEPSCs), it has the opposite effect at inhibitory synapses, decreasing their size and the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Contrary to CYFIP1 upregulation, its loss in vivo , upon conditional knockout in neocortical principal cells, increases expression of postsynaptic GABA A receptor β2/3-subunits and neuroligin 3, enhancing synaptic inhibition. Thus, CYFIP1 dosage can bi-directionally impact inhibitory synaptic structure and function, potentially leading to altered E/I balance and circuit dysfunction in CYFIP1-associated neurological disorders.
Peroxisomes are essential for a number of cellular functions, including reactive oxygen species metabolism, fatty acid b-oxidation and lipid synthesis. To ensure optimal functionality, peroxisomal size, shape and number must be dynamically maintained; however, many aspects of how this is regulated remain poorly characterised. Here, we show that the localisation of Miro1 and Miro2-outer mitochondrial membrane proteins essential for mitochondrial trafficking-to peroxisomes is not required for basal peroxisomal distribution and long-range trafficking, but rather for the maintenance of peroxisomal size and morphology through peroxisomal fission. Mechanistically, this is achieved by Miro negatively regulating Drp1-dependent fission, a function that is shared with the mitochondria. We further find that the peroxisomal localisation of Miro is regulated by its first GTPase domain and is mediated by an interaction through its transmembrane domain with the peroxisomal-membrane protein chaperone, Pex19. Our work highlights a shared regulatory role of Miro in maintaining the morphology of both peroxisomes and mitochondria, supporting a crosstalk between peroxisomal and mitochondrial biology.
It is well understood that apicomplexan parasites, such as the malaria pathogen Plasmodium, are descended from free-living algae, and maintain a vestigial chloroplast that has secondarily lost all genes of photosynthetic function. Recently, two fully photosynthetic relatives of parasitic apicomplexans have been identified, the ‘chromerid’ algae Chromera velia and Vitrella brassicaformis, which retain photosynthesis genes within their chloroplasts. Elucidating the processes governing gene expression in chromerid chloroplasts might provide valuable insights into the origins of parasitism in the apicomplexans. We have characterised chloroplast transcript processing pathways in C. velia, V. brassicaformis and P. falciparum with a focus on the addition of an unusual, 3′ poly(U) tail. We demonstrate that poly(U) tails in chromerids are preferentially added to transcripts that encode proteins that are directly involved in photosynthetic electron transfer, over transcripts for proteins that are not involved in photosynthesis. To our knowledge, this represents the first chloroplast transcript processing pathway to be associated with a particular functional category of genes. In contrast, Plasmodium chloroplast transcripts are not polyuridylylated. We additionally present evidence that poly(U) tail addition in chromerids is involved in the alternative processing of polycistronic precursors covering multiple photosynthesis genes, and appears to be associated with high levels of transcript abundance. We propose that changes to the chloroplast transcript processing machinery were an important step in the loss of photosynthesis in ancestors of parasitic apicomplexans.
Liver disease represents an increasing cause of global morbidity and mortality. Currently, liver transplant is the only treatment curative for end-stage liver disease. Donor organs cannot meet the demand and therefore scalable treatments and new disease models are required to improve clinical intervention. Pluripotent stem cells represent a renewable source of human tissue. Recent advances in three-dimensional cell culture have provided the field with more complex systems that better mimic liver physiology and function. Despite these improvements, current cell-based models are variable in performance and expensive to manufacture at scale. This is due, in part, to the use of poorly defined or cross-species materials within the process, severely affecting technology translation. To address this issue, we have developed an automated and economical platform to produce liver tissue at scale for modelling disease and small molecule screening. Stem cell derived liver spheres were formed by combining hepatic progenitors with endothelial cells and stellate cells, in the ratios found within the liver. The resulting tissue permitted the study of human liver biology ‘in the dish‘ and could be scaled for screening. In summary, we have developed an automated differentiation system that permits reliable self-assembly of human liver tissue for biomedical application. Going forward we believe that this technology will not only serve as an in vitro resource, and may have an important role to play in supporting failing liver function in humans.
Dissemination of malignant cells from primary tumours to metastatic sites is a key step in cancer progression. Disseminated tumour cells preferentially settle in specific target organs, and the success of such metastases depends on dynamic interactions between cancer cells and the microenvironments they encounter at secondary sites. Two emerging concepts concerning the biology of metastasis are that organ-specific microenvironments influence the fate of disseminated cancer cells, and that cancer cell-extracellular matrix interactions have important roles at all stages of the metastatic cascade. The extracellular matrix is the complex and dynamic non-cellular component of tissues that provides a physical scaffold and conveys essential adhesive and paracrine signals for a tissue's function. Here, we focus on how extracellular matrix dynamics contribute to liver metastases – a common and deadly event. We discuss how matrix components of the healthy and premetastatic liver support early seeding of disseminated cancer cells, and how the matrix derived from both cancer and liver contributes to the changes in niche composition as metastasis progresses. We also highlight the technical developments that are providing new insights into the stochastic, dynamic and multifaceted roles of the liver extracellular matrix in permitting and sustaining metastasis. An understanding of the contribution of the extracellular matrix to different stages of metastasis may well pave the way to targeted and effective therapies against metastatic disease.
Pancreatic cancer is a deadly and highly metastatic disease, although how metastatic lesions establish is not fully understood. A key feature of pancreatic tumours is extensive fibrosis and deposition of extracellular matrix (ECM). While pancreatic cancer cells are programmed by stimuli derived from a stiff ECM, metastasis requires loss of attachment and adaptation to a softer microenvironment at distant sites. Growing evidence suggests that stiff ECM influences pancreatic cancer cell behaviour. Here, we argue that this influence is reversible and that pancreatic cancer cells can be reprogrammed upon sensing soft substrates. Using engineered polyacrylamide hydrogels with tuneable mechanical properties, we show that collagen VI is specifically upregulated in pancreatic cancer cells on soft substrates, due to a lack of integrin engagement. Furthermore, the expression of collagen VI is inversely correlated with mechanosensing and activity of YAP (also known as YAP1), which might be due to a direct or indirect effect on transcription of genes encoding collagen VI. Collagen VI supports migration in vitro and metastasis formation in vivo. Metastatic nodules formed by pancreatic cancer cells lacking Col6a1 display stromal cell-derived collagen VI deposition, suggesting that collagen VI derived from either cancer cells or the stroma is an essential component of the metastatic niche. This article has an associated First Person interview with Vasileios Papalazarou, joint first author of the paper.
Adaptor protein (AP) complexes have critical roles in transmembrane protein sorting. AP-4 remains poorly understood in the brain despite its loss of function leading to a hereditary spastic paraplegia termed AP-4 deficiency syndrome. Here we demonstrate that knockout (KO) of AP-4 in a mouse model leads to thinning of the corpus callosum and ventricular enlargement, anatomical defects previously described in patients. At the cellular level, we find that AP-4 KO leads to defects in axonal extension and branching, in addition to aberrant distal swellings. Interestingly, we show that ATG9A, a key protein in autophagosome maturation, is critically dependent on AP-4 for its sorting from the trans-golgi network.Failure of AP-4 mediated ATG9A sorting results in its dramatic retention in the trans-golgi network in vitro and in vivo leading to a specific reduction of the axonal pool of ATG9A. As a result, autophagosome biogenesis is aberrant in the axon of AP-4 deficient neurons. The specific alteration to axonal integrity and axonal autophagosome maturation in AP-4 knockout neurons may underpin the pathology of AP-4 deficiency.
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