Leucine-rich repeat kinase 2 (LRRK2) not only plays a vital role in familial forms of Parkinson's disease (PD) but also represents a risk factor for idiopathic PD. Its multi-domain architecture enables fine-tuned regulation of its biological function by orchestrating intra- and inter-molecular interactions. Here, we present BioID proximity proteomes of LRRK2 that reveal new interactors, which we further characterize using a novel evolutionary and structural bioinformatics pipeline. Co-evolutionary analysis of the protein-protein interaction network identifies a structural and functional module enriched in cytoskeletal components associated with the centrosome and microtubules. In addition, structural modeling of binary interactions using AlphaFold-Multimer reveals distinct groups of interactors that engage LRRK2 in a manner dependent on specific conformations and epitopes. Furthermore, we identify distinct changes in the LRRK2 proximity proteome that are induced by the type I kinase inhibitor MLi-2 or by co-expression of the LRRK2 upstream effector RAB29. Depending on its activity state and conformation, these protein-protein interactions link LRRK2 to defined cellular sub-compartments, including centriolar satellites and vesicular sub-compartments.

Abstract The maintenance of protein homeostasis is vital for all cells. Alteration in protein handling underlies several diseases. The small molecule sephin1 is a promising clinical candidate against proteostasis disruption, but its mechanism of action is still uncertain. Our experimental evidence shows that sephin1 binds G-actin and drives actin cytoskeleton misfolding, and eventually, Golgi disintegration. At first, sephin1 impairs the autophagic flux and elicits the phosphorylation of the α subunit of eIF2 and the ER-stress independent expression of CHOP via GCN2 kinase. Sephin1 also inhibits the mammalian target of rapamycin (mTORC1), activates the transcription Factor EB (TFEB), drives the expression of TFEB-direct target genes, and eventually stimulates the autophagy lysosomal pathway. Our results reveal that the actin cytoskeleton may regulate autophagy via mTORC1-TFEB complemented with the GCN2-eIF2α-CHOP signaling pathway.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of late-onset familial and idiopathic Parkinson's disease (PD), known to date. Importantly, recent data from postmortem tissue as well as biomarker studies suggest that independent of mutations, increased kinase activity of LRRK2 plays an essential role in idiopathic PD pathogenesis. Despite extensive research on LRRK2, its activation mechanism(s) and how the various mutations result in increased kinase activity and neuronal death are still not completely understood. Accumulating evidence points to LRRK2 phosphoregulation, both autophosphorylation and phosphorylation by other kinases, as one potential molecular trigger of its activation. LRRK2 activation and localization are regulated by phosphatases such as protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A); however, the exact mechanism of this phosphoregulation is not known. Our data reveal that in vitro PP2A dephosphorylates sites within the RocCOR-GTPase domain of LRRK2 and, as a result, destabilizes LRRK2 dimers, with consequent reduction of its kinase activity. Strikingly, our data further highlight that LRRK2 in turn phosphorylates the catalytic subunit of the PP2A holoenzyme PPP2CA at its critical residue T304. Furthermore, LRRK2-mediated phosphorylation of PP2CA T304 alters the methylation of the C-terminus, which is crucial for both holoenzyme formation and catalytic activity. Importantly, expression of WT-PPP2CA protects from LRRK2-G2019S-induced neuronal cell death, while the PPP2CA-T304A mutant fails to do so, suggesting that impaired PP2A holoenzyme formation might be detrimental for LRRK2-PD.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of late-onset familial and idiopathic Parkinson’s disease (PD), known to date. Importantly, recent data from post-mortem tissue as well as biomarker studies suggest that independent of mutations, increased kinase activity of LRRK2 plays an essential role in idiopathic PD pathogenesis. Despite extensive research on LRRK2, its activation mechanism(s) and how the various mutations result in increased kinase activity and neuronal death is still not completely understood. Accumulating evidence points to LRRK2 phospho-regulation, both auto-phosphorylation and phosphorylation by other kinases, as one potential molecular trigger of its activation. LRRK2 activation and localization is regulated by phosphatases such as Protein phosphatase 1 (PP1) and Protein phosphatase 2A (PP2A), however the exact mechanism of this phospho-regulation is not known. Our data reveal that PP2A dephosphorylates sites within the RocCOR-GTPase domain of LRRK2 and as a result de-stabilizes LRRK2 dimers, with consequent reduction of its kinase activity. Strikingly, our data further highlight that LRRK2 in turn phosphorylates the catalytic subunit of the PP2A holoenzyme PPP2CA at its critical residue T304, crucial for both holoenzyme formation and catalytic activity. Importantly, expression of WT-PPP2CA protects from LRRK2-G2019S induced neuronal cell death, while PPP2CA-T304 mutants fail to do so, suggesting that impaired PP2A holoenzyme formation might be detrimental for LRRK2-PD. Significance statement Increased kinase activity of LRRK2 is a hallmark of Parkinson’s disease. Accumulating evidence points to LRRK2 phospho-regulation as one potential molecular trigger of its activation. However, the exact regulation of phosphorylation was not well understood. Our data reveal a feedback loop between LRRK2 and PP2A (a phosphatase) activity, which explains the neuroprotective properties of PP2A’s enzymatic activity in LRRK2-induced parkinsonism. Interestingly, PP2A activity is downregulated in alpha-synuclein PD models, having the same net-effect on PP2A activity as with hyper-active mutant LRRK2, and the methylation status of the C-terminal of PPP2CA plays an essential role in this regulation and progression of the disease. Understanding the regulation of PP2A, by post-translational modifications such as phosphorylation and methylation, can thus give important insight into the onset and progression of PD in general. ### Competing Interest Statement The authors have declared no competing interest. Michael J. Fox Foundation, 6709.3.

RAB3A encodes a small GTP-binding protein that is abundant in brain synaptic vesicles and crucial for the release of neurotransmitters and synaptic plasticity. Here, we identified RAB3A as a candidate gene for autosomal dominant cerebellar ataxia by two independent approaches: linkage in a large dominant ataxia family and, in parallel, an untargeted computational genetic association approach, analysing the 100 000 Genomes Project datasets. To validate the role of RAB3A in ataxia, we next screened large rare disease databases for rare heterozygous RAB3A variants in probands with ataxia features. In total, we identified 18 individuals from 10 unrelated families all sharing a cerebellar ataxia phenotype. Notably, 9 of the 10 families carried a recurrent variant in RAB3A, p.Arg83Trp, including one de novo occurrence. In addition, our screening revealed three families with a neurodevelopmental phenotype and three unique RAB3A variants, which were either de novo or loss-of-function variants. In line with the different RAB3A variant types, protein domains and predicted functional consequences, a comprehensive set of complementary methods was used to characterize the identified variants functionally. As expected, GTPase-activating protein (GAP)-dependent GTP hydrolysis was reduced for those two missense variants located in the GAP-binding domain of RAB3A (Arg83Trp and Tyr91Cys). In a Drosophila Rab3 loss-of-function model, these two missense variants also failed to rescue a synaptic phenotype. Overexpression of Rab3 variants in Drosophila wild-type background did not cause an obvious phenotype, making a dominant negative effect of these variants unlikely. Lastly, exploring interactors of RAB3A variants by using co-immunoprecipitation and mass spectrometry showed differential changes in variant-specific interactions with known RAB3A key regulatory and effector proteins. In sum, our results establish RAB3A as a neurological disease gene. It represents an autosomal dominant gene for cerebellar ataxia with different variants associated with disease, including the frequent reoccurring variant p.Arg83Trp. Our study sheds light on the variant-specific interactome of RAB3A. Finally, we suggest an association of RAB3A with a neurodevelopmental phenotype, as reported for variants in several RAB3A interaction partners and as seen in Rab3A-deficent mice, although this possible association warrants further investigation by future studies.

Cell-free microRNAs in body fluids have emerged as promising biomarker candidates in neurodegenerative diseases. While several studies have identified dysregulated miRNAs in sporadic Parkinson’s disease, it remains unclear whether distinguishable alterations of cell-free miRNAs occur in genetic forms of the disease, such as those associated with the LRRK2 G2019S mutation. In this proof-of-concept study, we used a human induced pluripotent stem cell-derived dopaminergic neuron model to investigate whether the LRRK2 G2019S mutation induces detectable changes in the intra- and extracellular miRNAome, and whether miRNA signatures identified in vitro can be validated in patient-derived cerebrospinal fluid. We differentiated dopaminergic neurons from induced pluripotent stem cells carrying the LRRK2 G2019S mutation and an isogenic gene-corrected control. Extracellular vesicles were isolated from the culture medium and used as a source of cell-free miRNA. Next, small RNA libraries were generated and analyzed. Differentially expressed microRNAs were validated in an independent batch using RT-qPCR. We further quantified candidate microRNAs in cerebrospinal fluid samples from five LRRK2 G2019S patients and matching healthy controls. The patient cohort included the fibroblast donor from whom the stem cells were originally derived. We successfully isolated extracellular vesicles from induced pluripotent stem cell-derived human dopaminergic neurons. We identified a distinct set of differentially expressed miRNAs in cellular and cell-free RNA, among which let-7g-5p and miR-21-5p were consistently upregulated and validated across independent replicates. These alterations were reflected in the cerebrospinal fluid of the original donor and partially reproduced in additional LRRK2 patients, supporting the concept of patient-specific signatures. A strong correlation between intra- and extracellular miRNA expression was observed. Our findings demonstrate that induced pluripotent stem cell-derived dopaminergic neurons can serve as a model to identify individualized, cell-free microRNA signatures associated with the LRRK2 G2019S mutation. The dysregulated miRNAs detected in vitro were mirrored in patient cerebrospinal fluid, supporting their potential as accessible molecular readouts. These results lay the groundwork for personalized biomarker strategies in genetic forms of Parkinson’s disease and warrant further validation in larger patient cohorts.

Mitochondrial markers help stratify Parkinson’s disease (PD) patients. We use high-throughput blotting to quantify Miro1, Mfn2, and VDAC levels in fibroblasts, blood cells, and iPSC-derived neurons. Miro1 is specifically retained in PD cells but degraded in healthy ones after mitochondrial depolarization. We correlate Miro1 retention scores with pathogenic mutations, genetic background, age, and clinical data. This scalable assay and quantifiable score for mitochondrial-PD support biomarker development and pharmacological screening.

Roco proteins entered the limelight after mutations in human LRRK2 were identified as a major cause of familial Parkinson’s disease. LRRK2 is a large and complex protein combining a GTPase and protein kinase activity, and disease mutations increase the kinase activity, while presumably decreasing the GTPase activity. Although a cross-communication between both catalytic activities has been suggested, the underlying mechanisms and the regulatory role of the GTPase domain remain unknown. Several structures of LRRK2 have been reported, but structures of Roco proteins in their activated GTP-bound state are lacking. Here, we use single-particle cryo-electron microscopy to solve the structure of a bacterial Roco protein (CtRoco) in its GTP-bound state, aided by two conformation-specific nanobodies: Nb Roco1 and Nb Roco2 . This structure presents CtRoco in an active monomeric state, featuring a very large GTP-induced conformational change using the LRR-Roc linker as a hinge. Furthermore, this structure shows how Nb Roco1 and Nb Roco2 collaborate to activate CtRoco in an allosteric way. Altogether, our data provide important new insights into the activation mechanism of Roco proteins, with relevance to LRRK2 regulation, and suggest new routes for the allosteric modulation of their GTPase activity.

Background Extracellular vesicles are easily accessible in various biofluids and allow the assessment of disease-related changes in the proteome. This has made them a promising target for biomarker studies, especially in the field of neurodegeneration where access to diseased tissue is very limited. Genetic variants in the LRRK2 gene have been linked to both familial and sporadic forms of Parkinson's disease. With LRRK2 inhibitors entering clinical trials, there is an unmet need for biomarkers that reflect LRRK2-specific pathology and target engagement.Methods In this study, we used induced pluripotent stem cells derived from a patient with Parkinson's disease carrying the LRRK2 G2019S mutation and an isogenic gene-corrected control to generate human dopaminergic neurons. We isolated extracellular vesicles and neuronal cell lysates and characterized their proteomic signature using data-independent acquisition proteomics. Then, we performed differential expression analysis to identify dysregulated proteins in the mutated line. We used Metascape and gene ontology enrichment analysis on the dysregulated proteomes to identify changes in associated functional networks.Results We identified 595 significantly differentially regulated proteins in extracellular vesicles and 3,205 in cell lysates. We visualized functionally relevant protein-protein interaction networks and identified key regulators within the dysregulated proteomes. Using gene ontology, we found a close association with biological processes relevant to neurodegeneration and Parkinson's disease. Finally, we focused on proteins that were dysregulated in both the extracellular and cellular proteomes. We provide a list of ten biomarker candidates that are functionally relevant to neurodegeneration and linked to LRRK2-associated pathology, for example, the sonic hedgehog signaling molecule, a protein that has tightly been linked to LRRK2-related disruption of cilia function.Conclusion In conclusion, we characterized the cellular and extracellular proteome of dopaminergic neurons carrying the LRRK2 G2019S mutation and proposed an experimentally based list of biomarker candidates for future studies.

The Roco proteins are a family of GTPases, characterized by the conserved presence of a Roc-COR tandem domain. These proteins entered the limelight after mutations in human LRRK2 were identified as a major cause of familial Parkinson’s disease. LRRK2 is a large and complex protein combining a GTPase and protein kinase activity, and disease mutation increase the kinase activity, while presumably decreasing the GTPase activity. Although a cross-communication between both catalytic activities has been suggested, the underlying mechanisms and the regulatory role of the GTPase domain remain unknown. Recently, several structures of LRRK2 have been reported, but so far structures of Roco proteins in their activated GTP-bound state are lacking. Here, we use single particle cryo-EM to solve the structure of a simpler bacterial Roco protein (CtRoco) in its GTP-bound state, aided by the use of two conformation-specific nanobodies: Nb Roco1 and Nb Roco2 . This structure presents CtRoco in an active monomeric state, featuring very significant conformational changes compared to the previously solved nucleotide-free dimer structure. In particular, the structure shows a very large GTP-induced conformational change of the LRR domain, unleashing it from the Roc-COR domains, using the Roc-LRR linker as a hinge. Furthermore, this structure shows how Nb Roco1 and Nb Roco2 collaborate to activate CtRoco in an allosteric way. Altogether, our data provides important new insights in the activation mechanism of Roco proteins, with relevance to LRRK2 regulation, and suggest new routes for the allosteric modulation of their GTPase activity.

In this study we employed a data-driven approach to explore the evolutionary and genetic determinants of protein direct interactions and stable complex formation in the human proteome. We found that simple co-evolutionary and co-expression metrics are highly informative of direct interactions and stable complexes. We used this information to train supervised binary classifiers to predict interactions either directly involved in the formation of a complex (as annotated in IntAct) or forming stable complexes (from Complex Portal). In the former task, our model was able to discriminate direct interactions with an AUROC=0.813, while in the latter it discriminated interaction forming stable complexes with an AUROC=0.964. In both cases, our approach outperformed String, that we employed as a baseline. Feature importance analysis revealed different contributions to the prediction of these distinct interaction types. Co-evolutionary features, in particular those referred to protein domains involved in interaction interfaces, are more important to discriminate direct interactions. On the other hand, co-expression features contributed more to the prediction of stable complexes. From these pairwise predictions we generated a proteome-wide network that we clustered to assess the recovery of known complexes from Complex Portal within network communities. We were able to recover known complexes at a higher accuracy compared to other approaches. In conclusion, we propose a new method able to discriminate direct interactions as well as forming stable complexes. This method can be used to stratify molecular interaction networks, as well as to perform discovery of new functional complexes at a proteome-wide scale. ### Competing Interest Statement The authors have declared no competing interest.

The Leucine-rich repeat kinase 2 (LRRK2) not only plays a vital role in familial forms of Parkinson's disease (PD) but is also considered as a risk factor for idiopathic PD. For these reasons, LRRK2 is considered a promising drug target for PD treatment. Its multi-domain architecture enables a fine-tuned regulation of its biological function by orchestrating intra- and inter-molecular interactions. At the same time, it offers multiple targetable epitopes for fine-tuned modulation of its deregulated kinase activity caused by pathogenic risk variants. Here, we present BioID proximity proteomes of LRRK2 revealing new interactors, which we further characterized by a novel evolutionary and structural bioinformatics pipeline. Co-evolutionary analysis of the protein-protein interaction (PPI) network identified a structural and functional module enriched in cytoskeletal components linked to the centrosome and microtubules. Likely co-evolved with LRRK2 within this cluster is CYLD, a K-63 selective ubiquitin deubiquitinase which we found to stabilize LRRK2. Furthermore, structural prediction of binary interactions via AlphaFold-multimer revealed distinct groups of interactors engaging with LRRK2 dependent on specific conformations and epitopes. We found two distinct groups engaging with LRRK2 either in an N-terminal closed ("locked") or open ("unlocked") conformation, both of which are associated with specific structurally defined interfaces and biological processes. Furthermore, we identified distinct changes in the LRRK2 proximity proteome induced by the type I kinase inhibitor MLi-2 or by co-expression of the LRRK2 upstream effector RAB29. Dependent on its state of activity and conformation, these functional state-specific protein interactions link LRRK2 to distinct cellular sub-compartments, including centriolar satellites as well as vesicular sub-compartments. ### Competing Interest Statement The authors have declared no competing interest.

The Parkinson’s Disease (PD)-linked protein Leucine Rich Repeat Kinase 2 (LRRK2) consists of seven domains, including a kinase and a Roc G domain. Despite the availability of several high-resolution structures, the dynamic regulation of its unique intramolecular domain stack is nevertheless still not well understood. By in-depth biochemical analysis, assessing the Michaelis-Menten kinetics of the Roc G domain, we have confirmed that LRRK2 has similar to other Roco protein family members a K M value of LRRK2 that lays within the range of the physiological GTP concentrations within the cell. Furthermore, the R1441G PD variant located within a mutational hotspot in the Roc domain showed an increased catalytic efficiency. The most common PD variant G2019S, located in the kinase domain showed an increased K M and reduced catalytic efficiency, suggesting a negative feedback mechanism from the kinase domain to the G-domain. Auto-phosphorylation of the G1+2 residue (T1343) in the Roc P-loop motif is critical for this phosphoregulation of both the K M as well as the k cat values of the Roc-catalyzed GTP hydrolysis, most likely by changing the monomer-dimer equilibrium. Together our data reveal a novel intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase dependent mechanism. Interestingly, PD mutants differently change the kinetics of the GTPase cycle, which might in part explain the difference in penetrance of these mutations in PD patients.

Leucine-rich repeat kinase 2 (LRRK2) is a large, multi-domain protein which is associated with Parkinson’s disease. Although high-resolution structures of LRRK2 are available, little is known about the complex dynamics behind the inter-domain regulation of LRRK2 and its perturbation by pathogenic variants. Previous studies have demonstrated that LRRK2 goes through an oligomerization cycle at the membrane, however it remains unclear in which form it exerts its kinase activity. Moreover, the LRRK2 monomer-dimer equilibrium and associated functional implications at a molecular level also need further investigation. In the present work, we used a multi-faceted approach to better understand LRRK2 oligomerization and suggest a functional model of how LRRK2 interacts with its substrates. To this end, we combined nano differential scanning calorimetry and mass photometry with molecular modelling. The thermal analysis resulted in a multistep denaturation profile, elucidating novel insights into the composite structural organization of the multi-domain protein LRRK2. Furthermore, LRRK2 shows a remarkable thermal stability, confirming its oligomeric nature. By using mass photometry, we could observe a monomer-dimer equilibrium which is altered by R1441G, a pathogenic variant within the Roc-COR interface. Most importantly, we could demonstrate that autophosphorylation induces LRRK2 monomerization, indicating a novel intramolecular feedback mechanism. Finally, we investigated the interaction of LRRK2 with its substrate, RAB10 by integrative computational modelling. The resulting models suggest that the monomeric form of LRRK2 is the favored protein conformation for the interaction with its substrate, leading to an increasing interest in the monomer-dimer equilibrium as a possible intervention point for the pathology.

Missense mutations along the leucine-rich repeat kinase 2 (LRRK2) protein are a major contributor to Parkinson's Disease (PD), the second most commonly occurring neurodegenerative disorder worldwide. We recently reported the development of allosteric constrained peptide inhibitors that target and downregulate LRRK2 activity through disruption of LRRK2 dimerization. In this study, we designed doubly constrained peptides with the objective of inhibiting C-terminal of Roc (COR)–COR mediated dimerization at the LRRK2 dimer interface. We show that the doubly constrained peptides are cell-permeant, bind wild-type and pathogenic LRRK2, inhibit LRRK2 dimerization and kinase activity, and inhibit LRRK2-mediated neuronal apoptosis, and in contrast to ATP-competitive LRRK2 kinase inhibitors, they do not induce the mislocalization of LRRK2 to skein-like structures in cells. This work highlights the significance of COR-mediated dimerization in LRRK2 activity while also highlighting the use of doubly constrained peptides to stabilize discrete secondary structural folds within a peptide sequence.

Supplemental mass photomerty raw data used in Figure 3 of 'Guaitoli, G., Zhang, X., Saitta, F., Miglionico, P., Silbermann, L.M., Ho, F.Y., Zweydorf, F.v., Signorelli, M., Tych, K., Fessas, D., Raimondi, F., Kortholt, A., and Gloeckner, C.J. (2023). Biophysical analysis reveals autophosphorylation as an important negative regulator of LRRK2 dimerization. bioRxiv, 2023.2008.2011.549911 doi: 10.1101/2023.08.11.549911' Raw data Figure 3A: Mass photometry raw data for LRRK2 wild-type in presence of different G-nucleotides at different LRRK2 concentrations. Three biological replicates have been considered for the statistical analysis. Raw data Figure 3B: Mass photometry raw data for LRRK2 WT (left panel), LRRK2 WT + MLi-2 (middle panel) and kinase-dead LRRK2 (right panel) without and with ATP pre-incubation (-/+ ATP). Two biological and three technical replicates have been considered for the statistical analysis. Raw data Figure 3C: Mass photometry raw data for pathogenic LRRK2 variants in presence of different G-nucleotides. Two biological and two technical replicates have been considered for the statistical analysis.

Introduction: SARS-CoV-2 infected patients with cancer have a worse outcome including a significant higher mortality, compared to non-cancer patients. However, limited data are available regarding in-hospital mortality during the Omicron phase of the pandemic. Therefore, the aim of the study was the comparison of mortality in patients with history of cancer and patients with active cancer disease during the different phases of the COVID-19 pandemic, focusing on the current Omicron variant of concern. Methods: We conducted a multicenter, observational, epidemiological cohort study at 45 hospitals in Germany. Until July 20, 2022, all adult hospitalized SARS-CoV-2 positive patients were included. The primary endpoint was in-hospital mortality regarding cancer status (history of cancer and active cancer disease) and SARS-CoV-2 virus type. Results: From March 11, 2020, to July 20, 2022, a total of 27,490 adult SARS-CoV-2 positive patients were included in the study. 2,578 patients (9.4%) had diagnosis of cancer, of whom 1,065 (41.3%) had history of cancer, whereas 1,513 (58.7%) had active cancer disease. Overall 3,749 out of the total of 27,490 patients (13.6%) died during the hospital stay. Patients with active cancer disease had a significantly higher mortality compared to patients without cancer diagnosis, in both phases of the pandemic (wild-type to Delta: OR 1.940 [1.646–2.285]); Omicron: 2.864 [2.354–3.486]). After adjustment to co-variables, SARS-CoV-2 infected patients with active cancer disease had the highest risk for in-hospital mortality compared to the other groups, in both phases of the pandemic. Conclusion: The CORONA Germany study indicates that hospitalized patients with active cancer disease are at high risk of death during a SARS-CoV-2 infection. Mortality of patients with history of cancer improved to nearly the level of non-cancer patients during Omicron phase.

Trans-activation response DNA binding protein of 43 kDa (TDP-43) regulates a great variety of cellular processes in the nucleus and cytosol. In addition, a defined subset of neurodegenerative diseases is characterized by nuclear depletion of TDP-43 as well as cytosolic mislocalization and aggregation. To perform its diverse functions TDP-43 can associate with different ribonucleoprotein complexes. Combined with transcriptomics, MS interactome studies have unveiled associations between TDP-43 and the spliceosome machinery, polysomes and RNA granules. Moreover, the highly dynamic, low-valency interactions regulated by its low-complexity domain calls for innovative proximity labeling methodologies. In addition to protein partners, the analysis of post-translational modifications showed that they may play a role in the nucleocytoplasmic shuttling, RNA binding, liquid-liquid phase separation and protein aggregation of TDP-43. Here we review the various TDP-43 ribonucleoprotein complexes characterized so far, how they contribute to the diverse functions of TDP-43, and roles of post-translational modifications. Further understanding of the fluid dynamic properties of TDP-43 in ribonucleoprotein complexes, RNA granules, and self-assemblies will advance the understanding of RNA processing in cells and perhaps help to develop novel therapeutic approaches for TDPopathies.