Biology subjects

Failla, A. V.

Publications and source records attributed to Failla, A. V..

9 recordsLinked to original sources

bioRxiv2026

MINFLUX-nanoscopy of hNAIP/NLRC4 inflammasome activation in single human macrophages

The biochemical and structural basis of NAIP/NLRC4 inflammasome activation is well understood. Far less well known are the spatiotemporal processes within cells that play a role in the activation of the NAIP/NLRC4 inflammasome. We used super-resolution imaging technology, including MINFLUX nanoscopy, to investigate the human hNAIP/hNLRC4 inflammasome in primary human macrophages following the phagocytosis of a specific Yersinia enterocolitica strain. hNAIP, which senses components of the bacterial type III secretion system (T3SS) and activates NLRC4, was recruited to bacteria-containing phagosomes after they were disrupted by the T3SS. Disruption of a single phagosome within a macrophage was sufficient to cause the accumulation of NLRC4 and NLRP3 (a sensor of another inflammasome), as well as the adapter protein ASC, in a dense condensate known as a speck. This subsequently led to plasma membrane permeation and pyroptosis. It appeared that, with increasing infection time, clusters containing NLRC4 and NLRP3/NLRC4 gradually acquired ASC for speck formation. MINFLUX-nanoscopy was able to visualise hNAIP at disrupted phagosomes with nanometre resolution, and also revealed the number and organisation of NLRC4 inflammasome discs within a speck, as well as their hNAIP content, for the first time. Surprisingly, the number of NLRC4 inflammasome discs varied considerably, ranging from 35 to 249 per speck. Also, only a small fraction of these seemed to contain hNAIP. Our results significantly improve our understanding of the process by which the hNAIP/NLRC4 inflammasome is activated in macrophages that have ingested bacteria containing a T3SS.

cell biology↗

bioRxiv2025

STED-FCS in subdiffraction limit volumes reveals altered diffusion in live cell applications

Fluorescence correlation spectroscopy (FCS) is a widely established light microscopy technique for investigating physiological parameters such as diffusion states, particle numbers, and viscosity in biological samples. Combining FCS with stimulated emission depletion (STED-FCS) has enabled the investigation of molecular diffusion to sub diffraction-limited volumes. However, the full potential of STED-FCS for biomedical applications remains underexplored. Here, we present proof-of-principle studies with novel implementations of STED-FCS for investigating biological processes in living samples. Specifically, we demonstrate the impact of STED-FCS analyses in three distinct biomedical assays. Firstly, we prove that STED-FCS is capable of observing dynamic changes in autophagy-related protein microtubule-associated protein 1 light chain 3b (LC3b) along neuronal axons. Secondly, we show that STED-FCS can resolve alterations in the carbohydrate chain lengths of glycoproteins in melanoma cells. Finally, we demonstrate that STED-FCS can measure the reduced mobility of lipids within the plasma membrane of neuronal cells treated with the Alzheimers disease-associated, aggregation-prone toxic peptide amyloid-beta 1-42 (A{beta}42). We believe that this study will inspire researchers to utilize STED-FCS to address critical questions in their bio-imaging studies, particularly regarding the super-resolution assessment of dynamic processes in living cells.

biophysics↗

bioRxiv2025

Amphisome biogenesis couples synaptic autophagy to local protein synthesis

Synaptic neurotransmission imposes high demands on membrane turnover, metabolism, and the remodeling of presynaptic molecular composition. While the impact of autophagy on neurotransmission has been firmly established, evidence for activity-dependent synaptic induction of autophagy remains surprisingly limited. Here, we demonstrate that amphisomes containing BDNF/TrkB are formed at presynaptic boutons following sustained synaptic activation. Activity-dependent bulk endocytosis serves as a membrane source for amphisome biogenesis, while key autophagy proteins are recruited to the active zone, and autophagy initiation is triggered locally by the energy-sensing kinase AMPK. BDNF/TrkB-containing amphisomes contribute to the turnover of key presynaptic cytoskeletal proteins involved in synaptic vesicle clustering. The formation of amphisomes following sustained synaptic activity facilitates both the degradation of these proteins and their replenishment through local translation of their mRNAs at presynaptic boutons. We propose that activity-induced synaptic autophagy largely reflects amphisome formation, which in turn is required for the replacement of proteins within the local presynaptic cytomatrix.

neuroscience↗

bioRxiv2023

Ring Array Illumination Microscopy: Combination of Super-Resolution with Large Field of View Imaging and long Working Distances

Here we present a novel fluorescence microscopy concept which enables a direct integration of Super-Resolution Microscopy (SRM) approaches (SIM/Nanosizing, STED, SMLM, MINFLUX, SIMFLUX) into microscopy systems with working distances (WD) up to the multicentimeter range while still allowing nanometer scale resolution at selected sites. This becomes possible by a "synthetic aperture" illumination mode with multiple, constructively interfering excitation beams positioned in a "Ring-Array" arrangement around a beam free interior zone containing instrumentation involved in complementary imaging modes. The feasibility of such a direct correlative microscopy method is validated by extensive numerical simulations; on the basis of these calculations, experimental implementation options are discussed. Such "Ring Array" illumination modes may be useful for various correlative microscopy methods, such as a direct combination of correlative light and electron microscopy in the same device (dCLEM); or a direct combination of low NA/large field-of-view widefield microscopy and super-resolution of selected sites in the same device (direct Correlative Opical Microscopy/dCOLM). Ring-Array supported correlative microscopy modes will open novel imaging perspectives in a variety of disciplines, from material sciences to biomedical applications.

biophysics↗

bioRxiv2022

The cohesin subunit RAD21.2 functions as a recombination silencer of ribosomal DNA arrays

Introductory ParagraphIn many species, including Arabidopsis, heterochromatin often comprises repetitive DNA elements, such as arrays of ribosomal DNA (rDNA). Repetitive regions pose a risk in meiosis since recombination between them can lead to gross genomic rearrangements. However, meiotic recombination at rDNA arrays and other heterochromatic repeat regions is blocked by not well understood mechanisms. Here, we have identified RAD21.2, an -kleisin subunit of cohesin, as a repressor of meiotic recombination at the rDNA regions in Arabidopsis. We show that RAD21.2 co-localizes with heterochromatic factors and is specifically enriched at rDNA repeats, which are devoid of the meiosis specific -kleisin REC8, needed for recombination. Knocking down RAD21.2, we find that REC8 moves into the nucleolus organizing regions (NORs), where we see an increase of RAD51 recombinase foci numbers. Concomitantly, we find extensive rearrangements of the NORs and the offspring of these plants have large variation in rDNA copy numbers demonstrating that RAD21.2 is necessary for transgenerational genome stability. One-Sentence SummaryThe cohesin component RAD21.2 represses meiotic recombination and by that contributes to genome stability over generations.

cell biology↗

bioRxiv2021

Visualization of bacterial type 3 secretion system components down the molecular level by MINFLUX nanoscopy

Type 3 secretion systems (T3SS) are essential virulence factors of numerous bacterial pathogens and inject effector proteins into host cells. The needle-like T3SS machinery consists of more than 20 components, has a length of around 100 nm and a diameter of up to 30 nm according to EM studies. Its intrabacterial components are highly dynamic and in permanent exchange with other bacterial structures. Therefore, a temporally and spatially resolved visualization of the T3SS using fluorescence microscopy techniques has been challenging. In the present study, novel labeling strategies were combined with super-resolution microscopy such as STED, STORM and MINFLUX. MINFLUX nanoscopy allowed to visualize components of the T3SS machinery such as the dynamic sorting platform component YscL and the extrabacterial pore protein YopD at unprecedented resolutions. The presented results represent the basis for an in depth investigation of T3SS structure and function and therefore gain new insights into the infection process of human pathogens in order to develop novel treatment and prevention strategies.

microbiology↗

bioRxiv2019

BRAIN-DERIVED EXTRACELLULAR VESICLES ARE HIGHLY ENRICHED IN THE PRION PROTEIN AND ITS C1 FRAGMENT: RELEVANCE FOR CELLULAR UPTAKE AND IMPLICATIONS IN STROKE

Extracellular vesicles (EVs) are important means of intercellular communication and a potent tool for regenerative therapy. In ischemic stroke, transient blockage of a brain artery leads to a lack of glucose and oxygen in the affected brain tissue, provoking neuronal death by necrosis in the core of the ischemic region. The fate of neurons in the surrounding penumbra depends on the stimuli, including EVs, received during the following hours. A detailed characterization of such stimuli is crucial not only for understanding stroke pathophysiology but also for new therapeutic interventions. In the present study, we characterize the EVs in mouse brain under physiological conditions and 24h after induction of transient ischemia in mice. We show that, in steady-state conditions, microglia are the main source of small EVs (sEVs) whereas after ischemia, the main EV population originates from astrocytes. Moreover, sEVs presented high amounts of the prion protein (PrP) which were increased after stroke. Conspicuously, sEVs were particularly enriched in a truncated PrP fragment (PrP-C1). Because of similarities between PrP-C1 and certain viral surface proteins, we studied the cellular uptake of brain-derived sEVs from mice lacking (PrP-KO) or containing PrP (WT). We show that PrP-KO-EVs are rapidly taken up by neurons and colocalize with lysosomes. Although eventually WT-EVs are also found in lysosomes, the amount taken up by neurons is significantly higher for PrP-KO-EVs. Likewise, microglia and astrocytes were also engulfing PrP-KO-sEVs more efficiently than WT-sEVs. Our results provide information on the relative contribution of brain cell types to the sEV pool in mice and indicate that increased release of sEVs by astrocytes together with elevated levels of PrP in sEVs may play a role in intercellular communication at early stages after stroke. In addition, amounts of PrP (and probably PrP-C1) in brain sEVs seem to contribute to their cellular uptake.

neuroscience↗

bioRxiv2019

SIPA1L2 controls trafficking and signaling of TrkB-containing amphisomes at presynaptic terminals

Amphisomes are transient organelles that derive from fusion of autophagosomes with late endosomes. They rapidly transform into degradative autolysosomes, whereas non-degradative roles of the autophagic pathway have been barely described. Here we show that in neurons BDNF/TrkB receptor bearing Rab7 / Light chain 3 (LC3) - positive amphisomes signal at presynaptic boutons during retrograde trafficking to the soma. Local signaling and inward transport essentially require the Rap GTPase-activating (RapGAP) protein SIPA1L2, which directly binds to TrkB and Snapin to connect TrkB-containing amphisomes to dynein. Association with LC3 regulates the RapGAP activity of SIPA1L2 and thereby retrograde trafficking. Following induction of presynaptic plasticity amphisomes dissociate from dynein at boutons, and this enables local signaling and promotes transmitter release. Accordingly, sipa1l2 knockout mice show impaired BDNF-dependent presynaptic plasticity. Collectively, the data suggest that TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity.

neuroscience↗

bioRxiv2018

Visualization and regulation of translocons in Yersinia type III protein secretion machines during host cell infection

Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery - also named injectisome - assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their assembly have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the putatively operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology we provide novel information about the spatiotemporal organisation of T3SS translocons and their regulation by host cell factors.\n\nAuthor SummaryMany human, animal and plant pathogenic bacteria employ a molecular machine termed injectisome to inject their toxins into host cells. Because injectisomes are crucial for these bacterias infectious potential they have been considered as targets for antiinfective drugs. Injectisomes are highly similar between the different bacterial pathogens and most of their overall structure is well established at the molecular level. However, only little information is available for a central part of the injectisome named the translocon. This pore-like assembly integrates into host cell membranes and thereby serves as an entry gate for the bacterial toxins. We used state of the art fluorescence microscopy to watch translocons of the diarrheagenic pathogen Yersinia enterocolitica during infection of human host cells. Thereby we could for the first time - with fluorescence microscopy - visualize translocons connected to other parts of the injectisome. Furthermore, because translocons mark functional injectisomes we could obtain evidence that injectisomes only become active when the bacteria are almost completely enclosed by host cells. These findings provide a novel view on the organisation and regulation of bacterial translocons and may thus open up new strategies to block the function of infectious bacteria.

microbiology↗