@article{14316, abstract = {Clathrin-mediated vesicle trafficking plays central roles in post-Golgi transport. In yeast (Saccharomyces cerevisiae), the AP-1 complex and GGA adaptors are predicted to generate distinct transport vesicles at the trans-Golgi network (TGN), and the epsin-related proteins Ent3p and Ent5p (collectively Ent3p/5p) act as accessories for these adaptors. Recently, we showed that vesicle transport from the TGN is crucial for yeast Rab5 (Vps21p)-mediated endosome formation, and that Ent3p/5p are crucial for this process, whereas AP-1 and GGA adaptors are dispensable. However, these observations were incompatible with previous studies showing that these adaptors are required for Ent3p/5p recruitment to the TGN, and thus the overall mechanism responsible for regulation of Vps21p activity remains ambiguous. Here, we investigated the functional relationships between clathrin adaptors in post-Golgi-mediated Vps21p activation. We show that AP-1 disruption in the ent3Δ5Δ mutant impaired transport of the Vps21p guanine nucleotide exchange factor Vps9p transport to the Vps21p compartment and severely reduced Vps21p activity. Additionally, GGA adaptors, the phosphatidylinositol-4-kinase Pik1p and Rab11 GTPases Ypt31p and Ypt32p were found to have partially overlapping functions for recruitment of AP-1 and Ent3p/5p to the TGN. These findings suggest a distinct role of clathrin adaptors for Vps21p activation in the TGN–endosome trafficking pathway.}, author = {Nagano, Makoto and Aoshima, Kaito and Shimamura, Hiroki and Siekhaus, Daria E and Toshima, Junko Y. and Toshima, Jiro}, issn = {1477-9137}, journal = {Journal of Cell Science}, number = {17}, publisher = {The Company of Biologists}, title = {{Distinct role of TGN-resident clathrin adaptors for Vps21p activation in the TGN-endosome trafficking pathway}}, doi = {10.1242/jcs.261448}, volume = {136}, year = {2023}, } @article{13316, abstract = {Although budding yeast has been extensively used as a model organism for studying organelle functions and intracellular vesicle trafficking, whether it possesses an independent endocytic early/sorting compartment that sorts endocytic cargos to the endo-lysosomal pathway or the recycling pathway has long been unclear. The structure and properties of the endocytic early/sorting compartment differ significantly between organisms; in plant cells, the trans-Golgi network (TGN) serves this role, whereas in mammalian cells a separate intracellular structure performs this function. The yeast syntaxin homolog Tlg2p, widely localizing to the TGN and endosomal compartments, is presumed to act as a Q-SNARE for endocytic vesicles, but which compartment is the direct target for endocytic vesicles remained unanswered. Here we demonstrate by high-speed and high-resolution 4D imaging of fluorescently labeled endocytic cargos that the Tlg2p-residing compartment within the TGN functions as the early/sorting compartment. After arriving here, endocytic cargos are recycled to the plasma membrane or transported to the yeast Rab5-residing endosomal compartment through the pathway requiring the clathrin adaptors GGAs. Interestingly, Gga2p predominantly localizes at the Tlg2p-residing compartment, and the deletion of GGAs has little effect on another TGN region where Sec7p is present but suppresses dynamics of the Tlg2-residing early/sorting compartment, indicating that the Tlg2p- and Sec7p-residing regions are discrete entities in the mutant. Thus, the Tlg2p-residing region seems to serve as an early/sorting compartment and function independently of the Sec7p-residing region within the TGN.}, author = {Toshima, Junko Y. and Tsukahara, Ayana and Nagano, Makoto and Tojima, Takuro and Siekhaus, Daria E and Nakano, Akihiko and Toshima, Jiro}, issn = {2050-084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{The yeast endocytic early/sorting compartment exists as an independent sub-compartment within the trans-Golgi network}}, doi = {10.7554/eLife.84850}, volume = {12}, year = {2023}, } @article{10712, abstract = {Solute carriers are increasingly recognized as participating in a plethora of pathologies, including cancer. We describe here the involvement of the orphan solute carrier MFSD1 in the regulation of tumor cell migration. Loss of MFSD1 enabled higher levels of metastasis in a mouse model. We identified an increased migratory potential in MFSD1-/- tumor cells which was mediated by increased focal adhesion turn-over, reduced stability of mature inactive β1 integrin, and the resulting increased integrin activation index. We show that MFSD1 promoted recycling to the cell surface of endocytosed inactive β1 integrin and thereby protected β1 integrin from proteolytic degradation; this led to dampening of the integrin activation index. Furthermore, down-regulation of MFSD1 expression was observed during early steps of tumorigenesis and higher MFSD1 expression levels correlate with a better cancer patient prognosis. In sum, we describe a requirement for endolysosomal MFSD1 in efficient β1 integrin recycling to suppress tumor spread.}, author = {Roblek, Marko and Bicher, Julia and van Gogh, Merel and György, Attila and Seeböck, Rita and Szulc, Bozena and Damme, Markus and Olczak, Mariusz and Borsig, Lubor and Siekhaus, Daria E}, issn = {2234-943X}, journal = {Frontiers in Oncology}, publisher = {Frontiers}, title = {{The solute carrier MFSD1 decreases β1 integrin’s activation status and thus tumor metastasis}}, doi = {10.3389/fonc.2022.777634}, volume = {12}, year = {2022}, } @article{10714, abstract = {Ribosomal defects perturb stem cell differentiation, causing diseases called ribosomopathies. How ribosome levels control stem cell differentiation is not fully known. Here, we discovered three RNA helicases are required for ribosome biogenesis and for Drosophila oogenesis. Loss of these helicases, which we named Aramis, Athos and Porthos, lead to aberrant stabilization of p53, cell cycle arrest and stalled GSC differentiation. Unexpectedly, Aramis is required for efficient translation of a cohort of mRNAs containing a 5’-Terminal-Oligo-Pyrimidine (TOP)-motif, including mRNAs that encode ribosomal proteins and a conserved p53 inhibitor, Novel Nucleolar protein 1 (Non1). The TOP-motif co-regulates the translation of growth-related mRNAs in mammals. As in mammals, the La-related protein co-regulates the translation of TOP-motif containing RNAs during Drosophila oogenesis. Thus, a previously unappreciated TOP-motif in Drosophila responds to reduced ribosome biogenesis to co-regulate the translation of ribosomal proteins and a p53 repressor, thus coupling ribosome biogenesis to GSC differentiation.}, author = {Martin, Elliot T. and Blatt, Patrick and Ngyuen, Elaine and Lahr, Roni and Selvam, Sangeetha and Yoon, Hyun Ah M. and Pocchiari, Tyler and Emtenani, Shamsi and Siekhaus, Daria E and Berman, Andrea and Fuchs, Gabriele and Rangan, Prashanth}, issn = {1878-1551}, journal = {Developmental Cell}, number = {7}, pages = {883--900.e10}, publisher = {Elsevier}, title = {{A translation control module coordinates germline stem cell differentiation with ribosome biogenesis during Drosophila oogenesis}}, doi = {10.1016/j.devcel.2022.03.005}, volume = {57}, year = {2022}, } @article{10713, abstract = {Cells migrate through crowded microenvironments within tissues during normal development, immune response, and cancer metastasis. Although migration through pores and tracks in the extracellular matrix (ECM) has been well studied, little is known about cellular traversal into confining cell-dense tissues. We find that embryonic tissue invasion by Drosophila macrophages requires division of an epithelial ectodermal cell at the site of entry. Dividing ectodermal cells disassemble ECM attachment formed by integrin-mediated focal adhesions next to mesodermal cells, allowing macrophages to move their nuclei ahead and invade between two immediately adjacent tissues. Invasion efficiency depends on division frequency, but reduction of adhesion strength allows macrophage entry independently of division. This work demonstrates that tissue dynamics can regulate cellular infiltration.}, author = {Akhmanova, Maria and Emtenani, Shamsi and Krueger, Daniel and György, Attila and Pereira Guarda, Mariana and Vlasov, Mikhail and Vlasov, Fedor and Akopian, Andrei and Ratheesh, Aparna and De Renzis, Stefano and Siekhaus, Daria E}, issn = {0036-8075}, journal = {Science}, number = {6591}, pages = {394--396}, publisher = {American Association for the Advancement of Science}, title = {{Cell division in tissues enables macrophage infiltration}}, doi = {10.1126/science.abj0425}, volume = {376}, year = {2022}, } @article{10918, abstract = {Cellular metabolism must adapt to changing demands to enable homeostasis. During immune responses or cancer metastasis, cells leading migration into challenging environments require an energy boost, but what controls this capacity is unclear. Here, we study a previously uncharacterized nuclear protein, Atossa (encoded by CG9005), which supports macrophage invasion into the germband of Drosophila by controlling cellular metabolism. First, nuclear Atossa increases mRNA levels of Porthos, a DEAD-box protein, and of two metabolic enzymes, lysine-α-ketoglutarate reductase (LKR/SDH) and NADPH glyoxylate reductase (GR/HPR), thus enhancing mitochondrial bioenergetics. Then Porthos supports ribosome assembly and thereby raises the translational efficiency of a subset of mRNAs, including those affecting mitochondrial functions, the electron transport chain, and metabolism. Mitochondrial respiration measurements, metabolomics, and live imaging indicate that Atossa and Porthos power up OxPhos and energy production to promote the forging of a path into tissues by leading macrophages. Since many crucial physiological responses require increases in mitochondrial energy output, this previously undescribed genetic program may modulate a wide range of cellular behaviors.}, author = {Emtenani, Shamsi and Martin, Elliot T and György, Attila and Bicher, Julia and Genger, Jakob-Wendelin and Köcher, Thomas and Akhmanova, Maria and Pereira Guarda, Mariana and Roblek, Marko and Bergthaler, Andreas and Hurd, Thomas R and Rangan, Prashanth and Siekhaus, Daria E}, issn = {1460-2075}, journal = {The Embo Journal}, publisher = {Embo Press}, title = {{Macrophage mitochondrial bioenergetics and tissue invasion are boosted by an Atossa-Porthos axis in Drosophila}}, doi = {10.15252/embj.2021109049}, volume = {41}, year = {2022}, } @article{12080, abstract = {Endocytosis is a multistep process involving the sequential recruitment and action of numerous proteins. This process can be divided into two phases: an early phase, in which sites of endocytosis are formed, and a late phase in which clathrin-coated vesicles are formed and internalized into the cytosol, but how these phases link to each other remains unclear. In this study, we demonstrate that anchoring the yeast Eps15-like protein Pan1p to the peroxisome triggers most of the events occurring during the late phase at the peroxisome. At this ectopic location, Pan1p recruits most proteins that function in the late phases—including actin nucleation promoting factors—and then initiates actin polymerization. Pan1p also recruited Prk1 kinase and actin depolymerizing factors, thereby triggering disassembly immediately after actin assembly and inducing dissociation of endocytic proteins from the peroxisome. These observations suggest that Pan1p is a key regulator for initiating, processing, and completing the late phase of endocytosis.}, author = {Enshoji, Mariko and Miyano, Yoshiko and Yoshida, Nao and Nagano, Makoto and Watanabe, Minami and Kunihiro, Mayumi and Siekhaus, Daria E and Toshima, Junko Y. and Toshima, Jiro}, issn = {1540-8140}, journal = {Journal of Cell Biology}, number = {10}, publisher = {Rockefeller University Press}, title = {{Eps15/Pan1p is a master regulator of the late stages of the endocytic pathway}}, doi = {10.1083/jcb.202112138}, volume = {221}, year = {2022}, } @article{10614, abstract = {The infiltration of immune cells into tissues underlies the establishment of tissue-resident macrophages and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. Here, we find that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio, which are themselves required for invasion. Both the filamin and the tetraspanin enhance the cortical activity of Rho1 and the formin Diaphanous and thus the assembly of cortical actin, which is a critical function since expressing a dominant active form of Diaphanous can rescue the Dfos macrophage invasion defect. In vivo imaging shows that Dfos enhances the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the properties of the macrophage nucleus from affecting tissue entry. We thus identify strengthening the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues. }, author = {Belyaeva, Vera and Wachner, Stephanie and György, Attila and Emtenani, Shamsi and Gridchyn, Igor and Akhmanova, Maria and Linder, M and Roblek, Marko and Sibilia, M and Siekhaus, Daria E}, issn = {1545-7885}, journal = {PLoS Biology}, number = {1}, pages = {e3001494}, publisher = {Public Library of Science}, title = {{Fos regulates macrophage infiltration against surrounding tissue resistance by a cortical actin-based mechanism in Drosophila}}, doi = {10.1371/journal.pbio.3001494}, volume = {20}, year = {2022}, } @phdthesis{11193, abstract = {The infiltration of immune cells into tissues underlies the establishment of tissue-resident macrophages and responses to infections and tumors. However, the mechanisms immune cells utilize to collectively migrate through tissue barriers in vivo are not yet well understood. In this thesis, I describe two mechanisms that Drosophila immune cells (hemocytes) use to overcome the tissue barrier of the germband in the embryo. One strategy is the strengthening of the actin cortex through developmentally controlled transcriptional regulation induced by the Drosophila proto-oncogene family member Dfos, which I show in Chapter 2. Dfos induces expression of the tetraspanin TM4SF and the filamin Cher leading to higher levels of the activated formin Dia at the cortex and increased cortical F-actin. This enhanced cortical strength allows hemocytes to overcome the physical resistance of the surrounding tissue and translocate their nucleus to move forward. This mechanism affects the speed of migration when hemocytes face a confined environment in vivo. Another aspect of the invasion process is the initial step of the leading hemocytes entering the tissue, which potentially guides the follower cells. In Chapter 3, I describe a novel subpopulation of hemocytes activated by BMP signaling prior to tissue invasion that leads penetration into the germband. Hemocytes that are deficient in BMP signaling activation show impaired persistence at the tissue entry, while their migration speed remains unaffected. This suggests that there might be different mechanisms controlling immune cell migration within the confined environment in vivo, one of these being the general ability to overcome the resistance of the surrounding tissue and another affecting the order of hemocytes that collectively invade the tissue in a stream of individual cells. Together, my findings provide deeper insights into transcriptional changes in immune cells that enable efficient tissue invasion and pave the way for future studies investigating the early colonization of tissues by macrophages in higher organisms. Moreover, they extend the current view of Drosophila immune cell heterogeneity and point toward a potentially conserved role for canonical BMP signaling in specifying immune cells that lead the migration of tissue resident macrophages during embryogenesis.}, author = {Wachner, Stephanie}, issn = {2663-337X}, pages = {170}, publisher = {Institute of Science and Technology Austria}, title = {{Transcriptional regulation by Dfos and BMP-signaling support tissue invasion of Drosophila immune cells}}, doi = {10.15479/at:ista:11193}, year = {2022}, } @article{9363, abstract = {Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson’s disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.}, author = {Inglés Prieto, Álvaro and Furthmann, Nikolas and Crossman, Samuel H. and Tichy, Alexandra Madelaine and Hoyer, Nina and Petersen, Meike and Zheden, Vanessa and Bicher, Julia and Gschaider-Reichhart, Eva and György, Attila and Siekhaus, Daria E and Soba, Peter and Winklhofer, Konstanze F. and Janovjak, Harald L}, issn = {15537404}, journal = {PLoS genetics}, number = {4}, pages = {e1009479}, publisher = {Public Library of Science}, title = {{Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease}}, doi = {10.1371/journal.pgen.1009479}, volume = {17}, year = {2021}, }