Biology subjects

Saftig, P.

Publications and source records attributed to Saftig, P..

11 recordsLinked to original sources

bioRxiv2026

TMEM55B promotes anterograde transport and distal axonal distribution of LAMP1-positive organelles

Lysosomal function throughout life is vital for the proper functioning and survival of post-mitotic neurons by mediating cellular signaling, degradation, and recycling processes. Their intracellular transport relies on microtubule-dependent mechanisms involving kinesin and dynein motors. While lysosomes were long thought absent from axons, recent studies have demonstrated the importance of LAMP1-positive endo-/lysosomal organelles in axonal compartments. This study investigates the physiological role of TMEM55B, a lysosomal membrane protein involved in lysosome positioning and motor coupling in neurons. TMEM55B is highly expressed in LAMP1-positive organelles in cortical neurons. We generated viable Tmem55b knockout mice that exhibited altered lysosomal positioning in neurons. Lysosomes clustered abnormally in the pre-axonal exclusion zone and the proximal axon, with many forming elongated tubular structures. Similarly, in primary rat neurons, TMEM55B depletion resulted in lysosomal tubulation and impaired bidirectional lysosomal transport, particularly in distal axon segments, without affecting lysosome velocity in the proximal and mid-axon. These transport defects were consistent with disrupted JIP4 function, a scaffold linking TMEM55B to both dynein and kinesin motors. TMEM55B deficiency did not result in neurodegeneration, behavioral deficits, or reduced lifespan in mice. As an orthogonal approach, we analyzed TMEM55B function in C. elegans. Worms lacking the TMEM55B homolog Y71H2AM.2 showed altered lysosomal dynamics in cholinergic neurons, supporting an evolutionarily conserved role. To test the involvement of TMEM55B's paralog, TMEM55A, in regulating neuronal lysosome positioning, we generated Tmem55a/b double-knockout mice, which exhibit impaired growth, premature death, and an overall aggravated phenotype. Collectively, our findings demonstrate that both TMEM55B and TMEM55A regulate lysosome distribution in neurons, particularly in axons, by influencing JIP4-dependent retro- and, notably, anterograde motor protein-mediated transport. This function is critical for spatial lysosomal homeostasis. Our study underscores the importance of TMEM55A/TMEM55B in regulating neuronal organelle dynamics and may be a factor considered in neurodegenerative diseases involving lysosomal trafficking defects.

cell biology↗

bioRxiv2026

ER-Lysosome Cholesterol Exchange Regulates Lysosomal Motility Through mTOR-Dependent LAMTOR1 Phosphorylation

The subcellular distribution of lysosomes, the main degradative organelles of mammalian cells, responds to metabolic cues in a highly dynamic way. While lysosomal positioning due to amino acid levels is well-characterized, cholesterol-dependent regulation of lysosomal motility is incompletely understood. We explored impaired lysosomal cholesterol export using a mass spectrometry-based multi-OMICs approach, identifying widespread reallocation of resources and signaling pathway modulation. We identified increased phosphorylation at LAMTOR1 serine 56 in response to cholesterol level perturbations. We demonstrate that this phosphorylation site is sufficient to disrupt Rag GTPases/SLC38A9 binding to the Ragulator complex, inhibiting canonical mTORC1 and facilitating binding of BORC, therefore promoting lysosomal retrograde movement. LAMTOR1 S56 phosphorylation responds exclusively to depletion of lysosomal limiting membrane cholesterol, is facilitated by mTOR, and presents a negative feedback loop for amino acid independent displacement of Ragulator bound Rag GTPases, limiting canonical mTORC1 activity. Mass spectrometry data are available via ProteomeXchange with identifier PXD073489. HighlightsO_LIPerturbation of lysosomal cholesterol homeostasis results in adaptation of cellular protein and lipid biosynthesis C_LIO_LILAMTOR1 is phosphorylated at serine 56 via mTORC1 C_LIO_LILAMTOR1 S56 phosphorylation is lysosomal membrane cholesterol dependent C_LIO_LILAMTOR1 S56 phosphorylation disrupts binding of Rag GTPases to the Ragulator complex C_LIO_LILAMTOR1 S56 phosphorylation promotes binding of Ragulator to BORC, facilitating lysosomal retrograde transport C_LI

molecular biology↗

bioRxiv2024

A non-catalytic function of a disintegrin and metalloprotease 10 determines hepatic progenitor cell fate

During chronic liver disease, hepatocytes may undergo proliferative arrest, leading to the activation, expansion and differentiation of hepatic progenitor cells (HPCs). Here we observe that expression of A Disintegrin And Metalloprotease (ADAM) 10 is increased in human and murine chronic liver disease correlating with HPC expansion. We report that proteolytic processing of ADAM10 by ADAM9 and generation of an ADAM10 intracellular domain that translocates to the nucleus, rather than ADAM10 enzymatic activity is essential for the regulation of HPC gene expression and differentiation. Genetic loss of ADAM10 in vitro and in vivo enhances stemness gene expression, increases the accumulation of undifferentiated HPCs and promotes the formation of liver fibrosis. Taken together, we demonstrate that a non-catalytic function of ADAM10 is an essential regulator of HPC fate and HPC-driven regeneration. Our data ascribe a non-proteolytic function to ADAM proteases which may be a general concept in adult tissue stem cells.

cell biology↗

bioRxiv2024

Tspan15 depletion in mice affects social investigation and aggressive behavior

Tetraspanins are transmembrane proteins that form tetraspanin-enriched microdomains at biological membranes and contribute to the formation of functional protein complexes involved in various cellular processes. Tspan7 and Tspan15 are highly expressed in the hippocampus, a brain region that contributes to the regulation of social aggression. While Tspan7 has been observed to modulate cognition and behavior in rodents, the function of Tspan15 in this context is still unknown. In this study, we tested preference for social novelty, territorial and aggressive behavior in mice lacking Tspan15 gene expression. We report that male Tspan15-KO mice exhibit an increased motivation for social investigation and show increased aggression compared to WT littermates, regardless of familiarity with the partner mouse. Our data suggest a possible role of Tspan15 as a molecular modulator of certain neuronal circuits that control aggressive behavior.

animal behavior and cognition↗

bioRxiv2024

Non-canonical function of ADAM10 in presynaptic plasticity

A Disintegrin And Metalloproteinase 10 (ADAM10) plays a pivotal role in shaping neuronal networks by orchestrating the activity of numerous membrane proteins through the shedding of their extracellular domains. Despite its significance in the brain, the specific cellular localization of ADAM10 remains not well understood due to a lack of appropriate tools. Here, using a specific ADAM10 antibody suitable for immunostainings, we discover that ADAM10 is localized to presynapses and especially enriched at presynaptic vesicles of mossy fiber (MF)-CA3 synapses in the hippocampus. These synapses undergo pronounced frequency facilitation of neurotransmitter release, a process that play critical roles in information transfer and neural computation. We demonstrate, that in conditional ADAM10 knockout mice the ability of MF synapses to undergo this type of synaptic plasticity is greatly reduced. The loss of facilitation depends on the cytosolic domain of ADAM10 and association with the calcium sensor synaptotagmin 7 rather than its proteolytic activity. Our findings unveil a new pathway contributing to the regulation of synaptic vesicle exocytosis.

neuroscience↗

bioRxiv2023

MFSD1 in complex with its accessory subunit GLMP functions as a general dipeptide uniporter in lysosomes

Lysosomal degradation of macromolecules in lysosomes produces diverse small metabolites exported by specific transporters for reuse in biosynthetic pathways. Here, we deorphanized the Major Facilitator Superfamily Domain Containing 1 (MFSD1) protein, which forms a tight complex with the Glycosylated Lysosomal Membrane Protein (GLMP) in the lysosomal membrane. Untargeted metabolomics analysis of MFSD1-deficient mouse lysosomes revealed an increase in cationic dipeptides. Purified MFSD1 selectively bound diverse dipeptides, while electrophysiological, isotope tracer, and fluorescence-based studies in Xenopus oocytes and proteoliposomes showed that MFSD1/GLMP acts as a uniporter for cationic and neutral dipeptides. Cryo-EM structure of the dipeptide-bound MFSD1/GLMP complex in outward-open conformation characterized the heterodimer interface and, in combination with molecular dynamics simulations, provided a structural basis for its selectivity towards diverse dipeptides. Together, our data identify MFSD1 as a general lysosomal dipeptide uniporter, providing an alternative route to recycle lysosomal proteolysis products when lysosomal amino acid exporters are overloaded.

biochemistry↗

bioRxiv2022

Nuclear envelope disruption triggers hallmarks of aging in lung alveolar macrophages

Aging is characterized by gradual immune dysfunction and increased risk for many diseases, including respiratory infections. Genomic instability is thought to play a central role in the aging process but the mechanisms that damage nuclear DNA in aging are insufficiently defined. Cells that migrate or reside within confined environments experience forces applied to their nucleus, leading to transient nuclear envelope (NE) ruptures. NE ruptures are associated with DNA damage, and Lamin A/C is required to limit these events. Here, we show that Lamin A/C protects lung alveolar macrophages from NE rupture and hallmarks of aging. Lamin A/C ablation in immune cells results in a selective depletion of lung alveolar macrophages (AM) and a heightened susceptibility to influenza infection. Lamin A/C-deficient AM that persist display constitutive nuclear envelope rupture marks, DNA damage and p53-dependent senescence. In wild-type mice, we found that AM migrate within constricted spaces in vivo, at heights that induce NE rupture and DNA damage. AM from aged wild-type mice and from Lamin A/C-deficient mice share an upregulated lysosomal signature with CD63 expression, and we find that CD63 is required to clear damaged DNA in macrophages. We propose that induction of genomic instability by NE disruption represents a mechanism of aging in alveolar macrophages.

immunology↗

bioRxiv2021

Lysosome-targeted lipid probes reveal sterol transporters NPC1 and LIMP2 as sphingosine transporters

Lysosomes are central catabolic organelles involved in lipid homeostasis and their dysfunction is associated with pathologies ranging from lysosomal storage disorders to common neurodegenerative diseases. The mechanism of lipid efflux from lysosomes is well understood for cholesterol, while the export of other lipids, particularly sphingosine, is less well studied. To overcome this knowledge gap, we have developed functionalized sphingosine and cholesterol probes that allow us to follow their metabolism, protein interactions as well as their subcellular localization. These probes feature a modified cage group for lysosomal targeting and controlled release of the active lipids with high temporal precision. An additional photo-crosslinkable group allowed for the discovery of lysosomal interactors for both sphingosine and cholesterol. In this way, we found that two lysosomal cholesterol transporters, NPC1 and LIMP-2/SCARB2, also directly bind to sphingosine. In addition, we showed that absence of either protein leads to lysosomal sphingosine accumulation which suggests a sphingosine transport role of both proteins. Furthermore, artificial elevation of lysosomal sphingosine levels impaired cholesterol efflux, consistent with sphingosine and cholesterol sharing a common export mechanism.

cell biology↗

bioRxiv2021

Ligands binding to the cellular prion protein induce its protective proteolytic release with therapeutic potential in neurodegenerative proteinopathies

The cellular prion protein (PrPC) is a central player in neurodegenerative diseases caused by protein misfolding, such as prion diseases or Alzheimers disease (AD). Expression levels of this GPI-anchored glycoprotein, especially at the neuronal cell surface, critically correlate with various pathomechanistic aspects underlying these diseases, such as templated misfolding (in prion diseases) and neurotoxicity and, hence, with disease progression and severity. In stark contrast to cell-associated PrPC, soluble extracellular forms or fragments of PrP are linked with neuroprotective effects, which is likely due to their ability to interfere with neurotoxic disease-associated protein conformers in the interstitial fluid. Fittingly, the endogenous proteolytic release of PrPC by the metalloprotease ADAM10 ( shedding) was characterized as a protective mechanism. Here, using a recently generated cleavage-site specific antibody, we shed new light on earlier studies by demonstrating that shed PrP (sPrP) negatively correlates with conformational conversion (in prion disease) and is markedly redistributed in murine brain in the presence of prion deposits or AD-associated amyloid plaques indicating a blocking and sequestrating activity. Importantly, we reveal that administration of certain PrP-directed antibodies and other ligands results in increased PrP shedding in cells and organotypic brain slice cultures. We also provide mechanistic and structural insight into this shedding-stimulating effect. In addition, we identified a striking exception to this, as one particular neuroprotective antibody, due to its special binding characteristics, did not cause increased shedding but rather strong surface clustering followed by fast endocytosis and degradation of PrPC. Both mechanisms may contribute to the beneficial action described for some PrP-directed antibodies/ligands and pave the way for new therapeutic strategies against devastating and currently incurable neurodegenerative diseases.

neuroscience↗

bioRxiv2021

LAMP3 is critical for surfactant homeostasis in mice

Lysosome-associated membrane glycoprotein 3 (LAMP3) is a type I transmembrane protein of the LAMP protein family with a cell-type-specific expression in alveolar type II cells in mice and hitherto unknown function. In type II pneumocytes, LAMP3 is localized in lamellar bodies, secretory organelles releasing pulmonary surfactant into the extracellular space to lower surface tension at the air/liquid interface. The physiological function of LAMP3, however, remains enigmatic. We generated Lamp3 knockout mice by CRISPR/Cas9. LAMP3 deficient mice are viable with an average life span and display regular lung function under basal conditions. The levels of a major hydrophobic protein component of pulmonary surfactant, SP-C, are strongly increased in the lung of Lamp3 knockout mice, and the lipid composition of the bronchoalveolar lavage shows mild but significant changes, resulting in alterations in surfactant functionality. In ovalbumin-induced experimental allergic asthma, the changes in lipid composition are aggravated, and LAMP3-deficient mice exert an increased airway resistance. Our data suggest a critical role of LAMP3 in the regulation of pulmonary surfactant homeostasis and normal lung function.

biochemistry↗

bioRxiv2019

Tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

A disintegrin and metalloprotease 10 (ADAM10) is essential for embryonic development and impacts on diseases such as cancer, Alzheimers and inflammatory diseases. ADAM10 is a molecular scissor that proteolytically cleaves the extracellular region from over 100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors and chemokines. ADAM10 was recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function is critically dependent on a TspanC8 partner. To address this, we generated the first monoclonal antibodies to Tspan15 as a model TspanC8. These were used to show that ADAM10 is the principal Tspan15-interacting protein, that Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Together these findings suggest that ADAM10 exists as an intimate ADAM10/TspanC8 scissor complex.

cell biology↗