Macromolecular Crowding, Phase Separation, and Homeostasis in the Orchestration of Bacterial Cellular Functions.

Macromolecular crowding affects the activity of proteins and functional macromolecular complexes in all cells, including bacteria. Crowding, together with physicochemical parameters such as pH, ionic strength, and the energy status, influences the structure of the cytoplasm and thereby indirectly macromolecular function. Notably, crowding also promotes the formation of biomolecular condensates by phase separation, initially identified in eukaryotic cells but more recently discovered to play key functions in bacteria. Bacterial cells require a variety of mechanisms to maintain physicochemical homeostasis, in particular in environments with fluctuating conditions, and the formation of biomolecular condensates is emerging as one such mechanism. In this work, we connect physicochemical homeostasis and macromolecular crowding with the formation and function of biomolecular condensates in the bacterial cell and compare the supramolecular structures found in bacteria with those of eukaryotic cells. We focus on the effects of crowding and phase separation on the control of bacterial chromosome replication, segregation, and cell division, and we discuss the contribution of biomolecular condensates to bacterial cell fitness and adaptation to environmental stress.

Rapid Optogenetic Clustering in the Cytoplasm with BcLOVclust.

Protein clustering is a powerful form of optogenetic control, yet remarkably few proteins are known to oligomerize with light. Recently, the photoreceptor BcLOV4 was found to form protein clusters in mammalian cells in response to blue light, although clustering coincided with its translocation to the plasma membrane, potentially constraining its application as an optogenetic clustering module. Herein we identify key amino acids that couple BcLOV4 clustering to membrane binding, allowing us to engineer a variant that clusters in the cytoplasm and does not associate with the membrane in response to blue light. This variant-called BcLOVclust-clustered over many cycles with substantially faster clustering and de-clustering kinetics compared to the widely used optogenetic clustering protein Cry2. The magnitude of clustering could be strengthened by appending an intrinsically disordered region from the fused in sarcoma (FUS) protein, or by selecting the appropriate fluorescent protein to which it was fused. Like wt BcLOV4, BcLOVclust activity was sensitive to temperature: light-induced clusters spontaneously dissolved at a rate that increased with temperature despite constant illumination. At low temperatures, BcLOVclust and Cry2 could be multiplexed in the same cells, allowing light control of independent protein condensates. BcLOVclust could also be applied to control signaling proteins and stress granules in mammalian cells. While its usage is currently best suited in cells and organisms that can be cultured below ∼30 °C, a deeper understanding of BcLOVclust thermal response will further enable its use at physiological mammalian temperatures.

Quantitative Analysis of Nanoplastics in Single Cells by Subcellular Chromatography.

The accumulation and spatial distribution of intracellular nanoplastic particles provide useful information about their spatiotemporal toxicological effects mediated by the physicochemical parameters of nanoplastics in living cells. In this study, a sample injection-transfer method was designed with an accuracy of up to femtoliters to attoliters to match the volume required for ultranarrow-bore open-tubular liquid chromatography. The separation and concentration quantification of mixed polystyrenes in different regions in living cells were achieved by directly transferring picoliter/femtoliter volumes of intracellular cytoplasm to an ultranarrow-bore open-tubular chromatographic column. The measurement of pollutant concentration in different areas of a small-volume target (single cell) was realized. This method is expected to be used in the qualitative and quantitative analyses of complex, mixed, and label-free nanoplastics (a few nm in size) in the subregions of living cells.

Physicochemical homeostasis in bacteria.

In living cells, the biochemical processes such as energy provision, molecule synthesis, gene expression, and cell division take place in a confined space where the internal chemical and physical conditions are different from those in dilute solutions. The concentrations of specific molecules and the specific reactions and interactions vary for different types of cells, but a number of factors are universal and kept within limits, which we refer to as physicochemical homeostasis. For instance, the internal pH of many cell types is kept within the range of 7.0 to 7.5, the fraction of macromolecules occupies 15%-20% of the cell volume (also known as macromolecular crowding) and the ionic strength is kept within limits to prevent salting-in or salting-out effects. In this article we summarize the generic physicochemical properties of the cytoplasm of bacteria, how they are connected to the energy status of the cell, and how they affect biological processes (Fig. 1). We describe how the internal pH and proton motive force are regulated, how the internal ionic strength is kept within limits, what the impact of macromolecular crowding is on the function of enzymes and the interaction between molecules, how cells regulate their volume (and turgor), and how the cytoplasm is structured. Physicochemical homeostasis is best understood in Escherichia coli, but pioneering studies have also been performed in lactic acid bacteria.

A general method for quantitative fractionation of mammalian cells.

Subcellular fractionation in combination with mass spectrometry-based proteomics is a powerful tool to study localization of key proteins in health and disease. Here we offered a reliable and rapid method for mammalian cell fractionation, tuned for such proteomic analyses. This method proves readily applicable to different cell lines in which all the cellular contents are accounted for, while maintaining nuclear and nuclear envelope integrity. We demonstrated the method's utility by quantifying the effects of a nuclear export inhibitor on nucleoplasmic and cytoplasmic proteomes.

The restriction of calcium influx in metaphase and post-metaphase stages of cell division revealed by imaging secondary ion mass spectrometry (SIMS).

A secondary ion mass spectrometry (SIMS)-based isotopic imaging technique of ion microscopy was used for observing calcium influx in single renal epithelial LLC-PK1 cells. The CAMECA IMS-3f SIMS instrument, used in the study, is capable of producing isotopic images of single cells at 500 nm spatial resolution. Due to the high-vacuum requirements of the instrument the cells were prepared cryogenically with a freeze-fracture method and frozen freeze-dried cells were used for SIMS analysis. The influx of calcium was imaged directly by exposure of cells to 44 Ca stable isotope in the extracellular buffer for 10 min. The 44 Ca influx was measured at mass 44 and the distribution of endogenous calcium at mass 40 (40 Ca) in the same cell. A direct comparison of interphase cells to cells undergoing division revealed that calcium influx is restricted in metaphase and post-metaphase stages of cell division. This restriction is lifted in late cytokinesis. The net influx of 44 Ca in 10 min was approximately half under calcium influx restriction in comparison to interphase cells. Under calcium influx restriction the 44 Ca concentration was the same between the mitotic chromosome and the cytoplasm. These observations indicate that the endoplasmic reticulum (ER) calcium uptake is compromised under calcium influx restriction in cells undergoing division.

Architecture of the cytoplasmic face of the nuclear pore.

INTRODUCTION The subcellular compartmentalization of eukaryotic cells requires selective transport of folded proteins and protein-nucleic acid complexes. Embedded in nuclear envelope pores, which are generated by the circumscribed fusion of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) are the sole bidirectional gateways for nucleocytoplasmic transport. The ~110-MDa human NPC is an ~1000-protein assembly that comprises multiple copies of ~34 different proteins, collectively termed nucleoporins. The symmetric core of the NPC is composed of an inner ring encircling the central transport channel and outer rings formed by Y­shaped coat nucleoporin complexes (CNCs) anchored atop both sides of the nuclear envelope. The outer rings are decorated with compartment­specific asymmetric nuclear basket and cytoplasmic filament nucleoporins, which establish transport directionality and provide docking sites for transport factors and the small guanosine triphosphatase Ran. The cytoplasmic filament nucleoporins also play an essential role in the irreversible remodeling of messenger ribonucleoprotein particles (mRNPs) as they exit the central transport channel. Unsurprisingly, the NPC's cytoplasmic face represents a hotspot for disease­associated mutations and is commonly targeted by viral virulence factors. RATIONALE Previous studies established a near-atomic composite structure of the human NPC's symmetric core by combining (i) biochemical reconstitution to elucidate the interaction network between symmetric nucleoporins, (ii) crystal and single-particle cryo-electron microscopy structure determination of nucleoporins and nucleoporin complexes to reveal their three-dimensional shape and the molecular details of their interactions, (iii) quantitative docking in cryo-electron tomography (cryo-ET) maps of the intact human NPC to uncover nucleoporin stoichiometry and positioning, and (iv) cell­based assays to validate the physiological relevance of the biochemical and structural findings. In this work, we extended our approach to the cytoplasmic filament nucleoporins to reveal the near-atomic architecture of the cytoplasmic face of the human NPC. RESULTS Using biochemical reconstitution, we elucidated the protein-protein and protein-RNA interaction networks of the human and Chaetomium thermophilum cytoplasmic filament nucleoporins, establishing an evolutionarily conserved heterohexameric cytoplasmic filament nucleoporin complex (CFNC) held together by a central heterotrimeric coiled­coil hub that tethers two separate mRNP­remodeling complexes. Further biochemical analysis and determination of a series of crystal structures revealed that the metazoan­specific cytoplasmic filament nucleoporin NUP358 is composed of 16 distinct domains, including an N­terminal S­shaped α­helical solenoid followed by a coiled­coil oligomerization element, numerous Ran­interacting domains, an E3 ligase domain, and a C­terminal prolyl­isomerase domain. Physiologically validated quantitative docking into cryo-ET maps of the intact human NPC revealed that pentameric NUP358 bundles, conjoined by the oligomerization element, are anchored through their N­terminal domains to the central stalk regions of the CNC, projecting flexibly attached domains as far as ~600 Å into the cytoplasm. Using cell­based assays, we demonstrated that NUP358 is dispensable for the architectural integrity of the assembled interphase NPC and RNA export but is required for efficient translation. After NUP358 assignment, the remaining 4-shaped cryo­ET density matched the dimensions of the CFNC coiled­coil hub, in close proximity to an outer-ring NUP93. Whereas the N-terminal NUP93 assembly sensor motif anchors the properly assembled related coiled­coil channel nucleoporin heterotrimer to the inner ring, biochemical reconstitution confirmed that the NUP93 assembly sensor is reused in anchoring the CFNC to the cytoplasmic face of the human NPC. By contrast, two C. thermophilum CFNCs are anchored by a divergent mechanism that involves assembly sensors located in unstructured portions of two CNC nucleoporins. Whereas unassigned cryo­ET density occupies the NUP358 and CFNC binding sites on the nuclear face, docking of the nuclear basket component ELYS established that the equivalent position on the cytoplasmic face is unoccupied, suggesting that mechanisms other than steric competition promote asymmetric distribution of nucleoporins. CONCLUSION We have substantially advanced the biochemical and structural characterization of the asymmetric nucleoporins' architecture and attachment at the cytoplasmic and nuclear faces of the NPC. Our near­atomic composite structure of the human NPC's cytoplasmic face provides a biochemical and structural framework for elucidating the molecular basis of mRNP remodeling, viral virulence factor interference with NPC function, and the underlying mechanisms of nucleoporin diseases at the cytoplasmic face of the NPC. [Figure: see text].

An intra-cytoplasmic route for SARS-CoV-2 transmission unveiled by Helium-ion microscopy.

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, the presence of: (1) long tunneling nanotubes that connect two or more host cells over submillimeter distances; (2) large scale multiple cell fusion events (syncytia); and (3) abundant extracellular vesicles of various sizes. Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.

A working model for condensate RNA-binding proteins as matchmakers for protein complex assembly.

Most cellular processes are carried out by protein complexes, but it is still largely unknown how the subunits of lowly expressed complexes find each other in the crowded cellular environment. Here, we will describe a working model where RNA-binding proteins in cytoplasmic condensates act as matchmakers between their bound proteins (called protein targets) and newly translated proteins of their RNA targets to promote their assembly into complexes. Different RNA-binding proteins act as scaffolds for various cytoplasmic condensates with several of them supporting translation. mRNAs and proteins are recruited into the cytoplasmic condensates through binding to specific domains in the RNA-binding proteins. Scaffold RNA-binding proteins have a high valency. In our model, they use homotypic interactions to assemble condensates and they use heterotypic interactions to recruit protein targets into the condensates. We propose that unoccupied binding sites in the scaffold RNA-binding proteins transiently retain recruited and newly translated proteins in the condensates, thus promoting their assembly into complexes. Taken together, we propose that lowly expressed subunits of protein complexes combine information in their mRNAs and proteins to colocalize in the cytoplasm. The efficiency of protein complex assembly is increased by transient entrapment accomplished by multivalent RNA-binding proteins within cytoplasmic condensates.

α-Synuclein interacts differently with membranes mimicking the inner and outer leaflets of neuronal membranes.

The toxicity of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease, is likely related to its interaction with the asymmetric neuronal membrane. α-Syn exists as cytoplasmatic and as extracellular protein as well. To shed light on the different interactions occurring at the different α-syn localizations, we have here modelled the external and internal membrane leaflets of the neuronal membrane with two complex lipid mixtures, characterized by phase coexistence and with negative charge confined to either the ordered or the disordered phase, respectively. To this purpose, we selected a five-component (DOPC/SM/DOPE/DOPS/chol) and a four-component (DOPC/SM/GM1/chol) lipid mixtures, which contained the main membrane lipid constituents and exhibited a phase separation with formation of ordered domains. We have compared the action of α-syn in monomeric form and at different concentrations (1 nM, 40 nM, and 200 nM) with respect to lipid systems with different composition and shape by AFM, QCM-D, and vesicle leakage experiments. The experiments coherently showed a higher stability of the membranes composed by the internal leaflet mixture to the interaction with α-syn. Damage to membranes made of the external leaflet mixture was detected in a concentration-dependent manner. Interestingly, the membrane damage was related to the fluidity of the lipid domains and not to the presence of negatively charged lipids.

PABPN1L assemble into "ring-like" aggregates in the cytoplasm of MII oocytes and is associated with female infertility†.

Infertility affects 10-15% of families worldwide. However, the pathogenesis of female infertility caused by abnormal early embryonic development is not clear. A recent study showed that poly(A)binding protein nuclear 1-like (PABPN1L) recruited BTG anti-proliferation factor 4 (BTG4) to mRNA 3'-poly(A) tails and was essential for maternal mRNA degradation. Here, we generated a PABPN1L-antibody and found "ring-like" PABPN1L aggregates in the cytoplasm of MII oocytes. PABPN1L-EGFP proteins spontaneously formed "ring-like" aggregates in vitro. This phenomenon is similar with CCR4-NOT catalytic subunit, CCR4-NOT transcription complex subunit 7 (CNOT7), when it starts deadenylation process in vitro. We constructed two mouse model (Pabpn1l-/- and Pabpn1l  tm1a/tm1a) simulating the intron 1-exon 2 abnormality of human PABPN1L and found that the female was sterile and the male was fertile. Using RNA-Seq, we observed a large-scale up-regulation of RNA in zygotes derived from Pabpn1l-/- MII oocytes. We found that 9222 genes were up-regulated instead of being degraded in the Pabpn1l-♀/+♂zygote. Both the Btg4 and CCR4-NOT transcription complex subunit 6 like (Cnot6l) genes are necessary for the deadenylation process and Pabpn1l-/- resembled both the Btg4 and Cnot6l knockouts, where 71.2% genes stabilized in the Btg4-♀/+♂ zygote and 84.2% genes stabilized in the Cnot6l-♀/+♂zygote were also stabilized in Pabpn1l-♀/+♂ zygote. BTG4/CNOT7/CNOT6L was partially co-located with PABPN1L in MII oocytes. The above results suggest that PABPN1L is widely associated with CCR4-NOT-mediated maternal mRNA degradation and PABPN1L variants on intron 1-exon 2 could be a genetic marker of female infertility.

The role of water coordination in the pH-dependent gating of hAQP10.

Human aquaporin 10 (hAQP10) is an aquaglyceroporin that assists in maintaining glycerol flux in adipocytes during lipolysis at low pH. Hence, a molecular understanding of the pH-sensitive glycerol conductance may open up for drug development in obesity and metabolically related disorders. Control of hAQP10-mediated glycerol flux has been linked to the cytoplasmic end of the channel, where a unique loop is regulated by the protonation status of histidine 80 (H80). Here, we performed unbiased molecular dynamics simulations of three protonation states of H80 to unravel channel gating. Strikingly, at neutral pH, we identified a water coordination pattern with an inverted orientation of the water molecules in vicinity of the loop. Protonation of H80 results in a more hydrophobic loop conformation, causing loss of water coordination and leaving the pore often dehydrated. Our results indicate that the loss of such water interaction network may be integral for the destabilization of the loop in the closed configuration at low pH. Additionally, a residue unique to hAQP10 (F85) reveals structural importance by flipping into the channel in correlation with loop movements, indicating a loop-stabilizing role in the closed configuration. Taken together, our simulations suggest a unique gating mechanism combining complex interaction networks between water molecules and protein residues at the loop interface. Considering the role of hAQP10 in adipocytes, the detailed molecular insights of pH-regulation presented here will help to understand glycerol pathways in these cells and may assist in drug discovery for better management of human adiposity and obesity.

A pre-screening strategy to assess resected tumor margins by imaging cytoplasmic viscosity and hypoxia.

To assure complete tumor removal, frozen section analysis is the most common procedure for intraoperative pathological assessment of resected tumor margins. However, during one operation, multiple biopsies may be sent for examination, but only few of them are made into cryosections because of the complex preparation protocols and time-consuming pathological analysis, which potentially increases the risk of overlooking tumor involvement. Here, we propose a fluorescence-based pre-screening strategy that allows high-throughput, convenient, and fast gross assessment of resected tumor margins. A dual-activatable cationic fluorescent molecular rotor was developed to specifically illuminate live tumor cells' cytoplasm by emitting two different fluorescence signals in response to elevations in hypoxia-induced nitroreductase (a biochemical marker) and cytoplasmic viscosity (a biophysical marker), two characteristics of cancer cells. The ability of the fluorescent molecular rotor in detecting tumor cells was evaluated in mouse and human specimens of multiple tissues by comparing with hematoxylin and eosin staining. Importantly, the fluorescent molecular rotor achieved 100 % specificity in discriminating lung and liver cancers from normal tissue, allowing pre-screening of the tumor-free surgical margins and promoting clinical decision. Altogether, this type of fluorescent molecular rotor and the proposed strategy may serve as a new option to facilitate intraoperative assessment of resected tumor margins.

Diversity in Sensing and Signaling of Bacterial Sensor Histidine Kinases.

Two-component signal transduction systems (TCSs) are widely conserved in bacteria to respond to and adapt to the changing environment. Since TCSs are also involved in controlling the expression of virulence, biofilm formation, quorum sensing, and antimicrobial resistance in pathogens, they serve as candidates for novel drug targets. TCSs consist of a sensor histidine kinase (HK) and its cognate response regulator (RR). Upon perception of a signal, HKs autophosphorylate their conserved histidine residues, followed by phosphotransfer to their partner RRs. The phosphorylated RRs mostly function as transcriptional regulators and control the expression of genes necessary for stress response. HKs sense their specific signals not only in their extracytoplasmic sensor domain but also in their cytoplasmic and transmembrane domains. The signals are sensed either directly or indirectly via cofactors and accessory proteins. Accumulating evidence shows that a single HK can sense and respond to multiple signals in different domains. The underlying molecular mechanisms of how HK activity is controlled by these signals have been extensively studied both biochemically and structurally. In this article, we introduce the wide diversity of signal perception in different domains of HKs, together with their recently clarified structures and molecular mechanisms.

The cellular environment shapes the nuclear pore complex architecture.

Nuclear pore complexes (NPCs) create large conduits for cargo transport between the nucleus and cytoplasm across the nuclear envelope (NE)1-3. These multi-megadalton structures are composed of about thirty different nucleoporins that are distributed in three main substructures (the inner, cytoplasmic and nucleoplasmic rings) around the central transport channel4-6. Here we use cryo-electron tomography on DLD-1 cells that were prepared using cryo-focused-ion-beam milling to generate a structural model for the human NPC in its native environment. We show that-compared with previous human NPC models obtained from purified NEs-the inner ring in our model is substantially wider; the volume of the central channel is increased by 75% and the nucleoplasmic and cytoplasmic rings are reorganized. Moreover, the NPC membrane exhibits asymmetry around the inner-ring complex. Using targeted degradation of Nup96, a scaffold nucleoporin of the cytoplasmic and nucleoplasmic rings, we observe the interdependence of each ring in modulating the central channel and maintaining membrane asymmetry. Our findings highlight the inherent flexibility of the NPC and suggest that the cellular environment has a considerable influence on NPC dimensions and architecture.

Influence of Incubation Time on Ortho-Toluidine Blue Mediated Antimicrobial Photodynamic Therapy Directed against Selected Candida Strains-An In Vitro Study.

(1) Background and the aim: The appropriate incubation time in the antimicrobial photodynamic therapy protocol seems to have a huge impact on the efficacy of this process. This is particularly important in relation to Candida strains, due to the size of these cells and the presence of the cell wall. The aims of this study were to determine the optimal incubation time needed for the absorption of toluidine blue by cells of C. albicans, C. glabrata, C. krusei and C. parapsilosis using direct observation by optical microscopy, and to evaluate the efficacy of TBO-mediated aPDT on planktonic cells of these strains. (2) Methods: The microscopic evaluation consisted of taking a series of images at a magnification of 600× and counting the % of stained cells. The in vitro effect of TBO-mediated aPDT combined with a diode laser (635 nm, 400mW, 12 J/cm2, CW) on the viability of yeast cells with different incubation times was evaluated. (3) Results: The presence of TBO within the cytoplasm was observed in all tested Candida strains and at all microscopic evaluation times. However, the highest percentages of cells were stained at 7 and 10 min. The highest % reduction of CFU/mL after TBO-mediated aPDT against Candida was obtained for the strain C. albicans ATCC 10,231 and it was 78.55%. (4) Conclusions: TBO-mediated aPDT against Candida was effective in reducing the number of CFU/mL at all assessed incubation times. However, the most efficient period for almost all strains was 7-10 min.

STING Signaling and Sterile Inflammation.

Innate immunity is regulated by a broad set of evolutionary conserved receptors to finely probe the local environment and maintain host integrity. Besides pathogen recognition through conserved motifs, several of these receptors also sense aberrant or misplaced self-molecules as a sign of perturbed homeostasis. Among them, self-nucleic acid sensing by the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway alerts on the presence of both exogenous and endogenous DNA in the cytoplasm. We review recent literature demonstrating that self-nucleic acid detection through the STING pathway is central to numerous processes, from cell physiology to sterile injury, auto-immunity and cancer. We address the role of STING in autoimmune diseases linked to dysfunctional DNAse or related to mutations in DNA sensing pathways. We expose the role of the cGAS/STING pathway in inflammatory diseases, neurodegenerative conditions and cancer. Connections between STING in various cell processes including autophagy and cell death are developed. Finally, we review proposed mechanisms to explain the sources of cytoplasmic DNA.

Unveiling mRNP composition by fluorescence correlation and cross-correlation spectroscopy using cell lysates.

Understanding the mRNA life cycle requires information about the dynamics and macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study these macromolecular structures because they have single molecule sensitivity and readily provide information about their molecular composition and dynamics. Here, we demonstrate how FCS can be exploited to study cytoplasmic mRNPs with high accuracy and reproducibility in cell lysates. Cellular lysates not only recapitulate data from live cells but provide improved readings and allow investigation of single mRNP analysis under particular conditions or following enzymatic treatments. Moreover, FCCS employing minute amounts of cells closely corroborated previously reported RNA dependent interactions and provided estimates of the relative overlap between factors in the mRNPs, thus depicting their heterogeneity. The described lysate-based FCS and FCCS analysis may not only complement current biochemical approaches but also provide novel opportunities for the quantitative analysis of the molecular composition and dynamics of single mRNPs.

SUMOylation Regulates TDP-43 Splicing Activity and Nucleocytoplasmic Distribution.

The nuclear RNA-binding protein TDP-43 forms abnormal cytoplasmic aggregates in the brains of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients and several molecular mechanisms promoting TDP-43 cytoplasmic mislocalization and aggregation have been proposed, including defects in nucleocytoplasmic transport, stress granules (SG) disassembly and post-translational modifications (PTM). SUMOylation is a PTM which regulates a variety of cellular processes and, similarly to ubiquitination, targets lysine residues. To investigate the possible regulatory effects of SUMOylation on TDP-43 activity and trafficking, we first assessed that TDP-43 is SUMO-conjugated in the nuclear compartment both covalently and non-covalently in the RRM1 domain at the predicted lysine 136 and SUMO-interacting motif (SIM, 106-110 residues), respectively. By using the SUMO-mutant TDP-43 K136R protein, we demonstrated that SUMOylation modifies TDP-43 splicing activity, specifically exon skipping, and influences its sub-cellular localization and recruitment to SG after oxidative stress. When promoting deSUMOylation by SENP1 enzyme over-expression or by treatment with the cell-permeable SENP1 peptide TS-1, the cytoplasmic localization of TDP-43 increased, depending on its SUMOylation. Moreover, deSUMOylation by TS-1 peptide favoured the formation of small cytoplasmic aggregates of the C-terminal TDP-43 fragment p35, still containing the SUMO lysine target 136, but had no effect on the already formed p25 aggregates. Our data suggest that TDP-43 can be post-translationally modified by SUMOylation which may regulate its splicing function and trafficking, indicating a novel and druggable mechanism to explore as its dysregulation may lead to TDP-43 pathological aggregation in ALS and FTD.