scholarly journals ATP-driven separation of liquid phase condensates in bacteria

2019 ◽  
Author(s):  
B. Guilhas ◽  
J.C. Walter ◽  
J. Rech ◽  
G. David ◽  
N.-O. Walliser ◽  
...  
Keyword(s):  
Liquid Phase ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Cellular Space ◽  
Dna Segregation ◽  
Active Mechanism

AbstractLiquid-liquid phase separated (LLPS) states are key to compartmentalise components in the absence of membranes, however it is unclear whether LLPS condensates are actively and specifically organized in the sub-cellular space and by which mechanisms. Here, we address this question by focusing on the ParABS DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB) and a motor (ParA). We show that parS-ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume, but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favoured by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates and localises non-canonical LLPS condensates in the sub-cellular space.

scholarly journals Aggresome Formation and Liquid-liquid Phase Separation Independently Induce Cytoplasmic Aggregation of TAR DNA-binding protein 43

2020 ◽  
Author(s):  
Koji Yamanaka ◽  
Seiji Watanabe ◽  
Hidekazu Inami ◽  
Kotaro Oiwa ◽  
Yuri Murata ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Liquid Phase Separation ◽  
Mammalian Expression ◽  
Sporadic Als ◽  
Aggresome Formation ◽  
Liquid Liquid Phase Separation

Abstract Background Cytoplasmic inclusion of TAR DNA-binding protein 43 (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and a subtype of frontotemporal lobar degeneration (FTLD). Recent studies have suggested that the formation of cytoplasmic TDP-43 aggregates is dependent on a liquid-liquid phase separation (LLPS) mechanism. However, it is unclear whether TDP-43 pathology is induced through a single intracellular mechanism such as LLPS.Methods We established a TDP-43 aggregation screening system using a cultured neuronal cell line stably expressing EGFP-fused TDP-43 and a mammalian expression library of the inherited ALS/FTLD causative genes. We performed a screening to identify the intracellular mechanisms responsible for TDP-43 aggregation. An immunofluorescence study was conducted using the sporadic ALS spinal cord sections.Results We found that microtubule-related proteins (MRPs) and RNA-binding proteins (RBPs) co-aggregated with TDP-43. MRPs and RBPs sequestered TDP-43 into the cytoplasmic aggregates through distinct mechanisms such as microtubules and LLPS, respectively. The MRPs-induced TDP-43 aggregates were co-localized with aggresomal markers and dependent on histone deacetylase 6 (HDAC6), suggesting that aggresome formation induced the co-aggregation. However, the MRPs-induced aggregates were not affected by 1,6-hexanediol, an LLPS inhibitor. On the other hand, the RBPs-induced TDP-43 aggregates were sensitive to 1,6-hexanediol, but not dependent on microtubules or HDAC6. In sporadic ALS patients, approximately half of skein-like TDP-43 inclusions were co-localized with HDAC6, but round and granular type inclusion were not. Moreover, HDAC6-positive and HDAC6-negative inclusions were found in the same ALS patient, suggesting that the two distinct pathways are both involved in TDP-43 pathology.Conclusion Our findings suggest that at least two distinct pathways (i.e., aggresome formation and LLPS ) are involved in inducing the TDP-43 pathologies.

scholarly journals Aggresome formation and liquid–liquid phase separation independently induce cytoplasmic aggregation of TAR DNA-binding protein 43

2020 ◽  
Vol 11 (10) ◽  
Author(s):  
Seiji Watanabe ◽  
Hidekazu Inami ◽  
Kotaro Oiwa ◽  
Yuri Murata ◽  
Shohei Sakai ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Liquid Phase Separation ◽  
Mammalian Expression ◽  
Sporadic Als ◽  
Aggresome Formation ◽  
Liquid Liquid Phase Separation

Abstract Cytoplasmic inclusion of TAR DNA-binding protein 43 (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and a subtype of frontotemporal lobar degeneration (FTLD). Recent studies have suggested that the formation of cytoplasmic TDP-43 aggregates is dependent on a liquid–liquid phase separation (LLPS) mechanism. However, it is unclear whether TDP-43 pathology is induced through a single intracellular mechanism such as LLPS. To identify intracellular mechanisms responsible for TDP-43 aggregation, we established a TDP-43 aggregation screening system using a cultured neuronal cell line stably expressing EGFP-fused TDP-43 and a mammalian expression library of the inherited ALS/FTLD causative genes, and performed a screening. We found that microtubule-related proteins (MRPs) and RNA-binding proteins (RBPs) co-aggregated with TDP-43. MRPs and RBPs sequestered TDP-43 into the cytoplasmic aggregates through distinct mechanisms, such as microtubules and LLPS, respectively. The MRPs-induced TDP-43 aggregates were co-localized with aggresomal markers and dependent on histone deacetylase 6 (HDAC6), suggesting that aggresome formation induced the co-aggregation. However, the MRPs-induced aggregates were not affected by 1,6-hexanediol, an LLPS inhibitor. On the other hand, the RBPs-induced TDP-43 aggregates were sensitive to 1,6-hexanediol, but not dependent on microtubules or HDAC6. In sporadic ALS patients, approximately half of skein-like TDP-43 inclusions were co-localized with HDAC6, but round and granular type inclusion were not. Moreover, HDAC6-positive and HDAC6-negative inclusions were found in the same ALS patient, suggesting that the two distinct pathways are both involved in TDP-43 pathology. Our findings suggest that at least two distinct pathways (i.e., aggresome formation and LLPS) are involved in inducing the TDP-43 pathologies.

The DNA-binding protein YB-1 is a direct MYCN target and influences repair and resistance in neuroblastoma cell lines

2010 ◽  
Vol 222 (03) ◽  
Author(s):  
S Degen ◽  
S Kuhfittig-Kulle ◽  
JH Schulte ◽  
F Westermann ◽  
A Schramm ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Lines ◽  
Binding Protein ◽  
Neuroblastoma Cell ◽  
Dna Binding Protein ◽  
Neuroblastoma Cell Lines
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