통찰 - Computational Biology - # Dynamics and Localization of Nuclear Pore Complexes

Stable Nuclear Basket Proteins Regulate the Distribution and Mobility of Nuclear Pore Complexes in Budding Yeast

Q: How do the biophysical properties of the nucleolus contribute to the exclusion of Mlp-positive NPCs from this region?

The biophysical properties of the nucleolus play a crucial role in the exclusion of Mlp-positive NPCs from this region. The nucleolus is a membrane-less organelle that forms through liquid-liquid phase separation, creating a distinct environment within the nucleus. This phase separation is driven by the interactions between nucleolar proteins and nucleic acids, leading to the formation of a dense, gel-like structure. The nucleolus has a higher viscosity compared to the surrounding nucleoplasm, which affects the diffusion of molecules within and around it. In the context of NPC exclusion, the biophysical properties of the nucleolus create a physical barrier that restricts the entry of Mlp-positive NPCs. The dense and viscous nature of the nucleolus hinders the movement of large protein complexes like NPCs, preventing their localization within this region. Additionally, the specific composition of the nucleolus, rich in ribosomal RNA and associated proteins, may not provide the necessary binding sites or interactions for Mlp-positive NPCs to anchor or remain stable within the nucleolar territory. Therefore, the biophysical properties of the nucleolus act as a spatial cue that guides the distribution of NPCs on the nuclear envelope, keeping Mlp-positive NPCs predominantly in the non-nucleolar territory.

Q: What are the specific chromatin interactions or other molecular mechanisms that mediate the preferential localization of NPCs to the non-nucleolar territory?

The preferential localization of NPCs to the non-nucleolar territory is mediated by specific chromatin interactions and other molecular mechanisms. One key player in this process is the nuclear basket proteins Mlp1 and Mlp2, which interact with chromatin and mRNA processing factors to regulate NPC distribution. These proteins form stable assemblies at the NPC and are responsible for excluding NPCs from the nucleolar region. The interaction of Mlp1 and Mlp2 with chromatin components and mRNA maturation factors helps anchor NPCs to specific regions of the nuclear envelope, preventing their entry into the nucleolar territory. Additionally, Nup2, another nucleoporin recruited to the NPC by Nup60, plays a role in restricting the localization of NPCs to the non-nucleolar territory. Nup2 interacts with chromatin and other nuclear components, influencing the mobility and distribution of NPCs on the nuclear envelope. The combined effects of Mlp1/2 and Nup2 create a dynamic network of interactions that regulate the spatial organization of NPCs within the nucleus. Other molecular mechanisms involved in mediating the preferential localization of NPCs to the non-nucleolar territory may include the binding of specific nuclear factors or signaling molecules that regulate NPC movement and anchoring. Further research is needed to fully elucidate the intricate network of interactions that govern NPC distribution in the nucleus.

Q: Can the doRITE and single NPC tracking approach be applied to study NPC organization and dynamics in other eukaryotic systems beyond budding yeast?

The doRITE and single NPC tracking approach developed in budding yeast can be adapted and applied to study NPC organization and dynamics in other eukaryotic systems. The principles of doRITE, which involve recombination-induced tag exchange to track individual NPCs through multiple cell cycles, can be implemented in other model organisms with stable protein complexes like NPCs. By tagging specific nucleoporins with fluorescent markers and inducing recombination, researchers can visualize and track the movement of individual NPCs in different cellular contexts. Single NPC tracking using advanced microscopy techniques can provide valuable insights into the dynamics of NPC assembly, stability, and interactions with other cellular components. This approach can be applied to diverse eukaryotic systems, including mammalian cells, Drosophila, and C. elegans, to investigate the spatial organization and regulation of NPCs in different cellular environments. By combining doRITE with single NPC tracking, researchers can uncover novel mechanisms governing NPC behavior and function across various organisms, enhancing our understanding of nuclear transport and organization.

핵심 개념

The nuclear basket proteins Mlp1, Mlp2, and Pml39 form a stable assembly on nuclear pore complexes (NPCs) and mediate their exclusion from the nucleolar territory, while Nup2 independently restricts the localization of NPCs to the non-nucleolar region.

초록

The study investigates the dynamics and localization of nuclear pore complexes (NPCs) in budding yeast using a novel technique called recombination-induced tag exchange (RITE), which allows visualization of individual NPCs.

Key highlights:

The nuclear basket proteins Mlp1, Mlp2, and Pml39 are stably associated with NPCs, contrary to previous reports of their dynamic behavior.
Mlp1 and Mlp2 are responsible for the exclusion of NPCs from the nucleolar territory of the nucleus.
Nup2 also contributes to the reduced density of NPCs in the nucleolar region through an independent pathway.
Tracking of individual NPCs reveals that the presence of Mlp1/2 reduces the mobility of NPCs, which likely contributes to their exclusion from the nucleolar territory.
The authors develop a method for single NPC tracking in budding yeast, providing a powerful tool to study NPC organization and dynamics.

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biorxiv.org

통계

The average number of Nup133-RITE(GFP-to-dark) and Nup188-RITE(GFP-to-dark) foci per cell decays exponentially, with an estimated 158 ± 22 NPCs per cell at the time of recombination induction.
The intensity of Mlp2-GFP and Pml39-GFP at the nuclear envelope is 45.3 ± 5% and 26.9 ± 7% of the Mlp1-GFP intensity, respectively.
Deletion of MLP1 and MLP2 increases the fraction of 14-hour old NPCs detected in the nucleolar territory from 10 ± 3% in wild type to 15 ± 2% and 19 ± 4%, respectively.
The ratio of NPC density in the nucleolar versus non-nucleolar territory is 0.53 ± 0.06 in wild type, but increases to 0.75 ± 0.07 in the absence of Mlp1 and Mlp2.
The average diffusion coefficients of NPCs are 63 ± 8% and 53 ± 14% higher in mlp1Δ mlp2Δ and nup60Δ mutants, respectively, compared to wild type.

인용구

"Mlp1, Mlp2 and Pml39 associate stably with NPCs over multiple generations, although we cannot exclude the existence of an additional more dynamic pool of these proteins which could not be visualized by doRITE."
"Nup60 is required for the assembly of Mlp1 into the NPC but not for its maintenance."
"Our data and simulation thus support the hypothesis that an increased apparent diffusion coefficient of NPCs on the nuclear envelope contributes to the reduced NPC density in the nucleolar territory."

핵심 통찰 요약

The nuclear basket regulates the distribution and mobility of nuclear pore complexes in budding yeast

by Zsok,J., Sim... 게시일 www.biorxiv.org 09-28-2023

https://www.biorxiv.org/content/10.1101/2023.09.28.558499v2

더 깊은 질문

How do the biophysical properties of the nucleolus contribute to the exclusion of Mlp-positive NPCs from this region?

The biophysical properties of the nucleolus play a crucial role in the exclusion of Mlp-positive NPCs from this region. The nucleolus is a membrane-less organelle that forms through liquid-liquid phase separation, creating a distinct environment within the nucleus. This phase separation is driven by the interactions between nucleolar proteins and nucleic acids, leading to the formation of a dense, gel-like structure. The nucleolus has a higher viscosity compared to the surrounding nucleoplasm, which affects the diffusion of molecules within and around it.
In the context of NPC exclusion, the biophysical properties of the nucleolus create a physical barrier that restricts the entry of Mlp-positive NPCs. The dense and viscous nature of the nucleolus hinders the movement of large protein complexes like NPCs, preventing their localization within this region. Additionally, the specific composition of the nucleolus, rich in ribosomal RNA and associated proteins, may not provide the necessary binding sites or interactions for Mlp-positive NPCs to anchor or remain stable within the nucleolar territory. Therefore, the biophysical properties of the nucleolus act as a spatial cue that guides the distribution of NPCs on the nuclear envelope, keeping Mlp-positive NPCs predominantly in the non-nucleolar territory.

What are the specific chromatin interactions or other molecular mechanisms that mediate the preferential localization of NPCs to the non-nucleolar territory?

The preferential localization of NPCs to the non-nucleolar territory is mediated by specific chromatin interactions and other molecular mechanisms. One key player in this process is the nuclear basket proteins Mlp1 and Mlp2, which interact with chromatin and mRNA processing factors to regulate NPC distribution. These proteins form stable assemblies at the NPC and are responsible for excluding NPCs from the nucleolar region. The interaction of Mlp1 and Mlp2 with chromatin components and mRNA maturation factors helps anchor NPCs to specific regions of the nuclear envelope, preventing their entry into the nucleolar territory.
Additionally, Nup2, another nucleoporin recruited to the NPC by Nup60, plays a role in restricting the localization of NPCs to the non-nucleolar territory. Nup2 interacts with chromatin and other nuclear components, influencing the mobility and distribution of NPCs on the nuclear envelope. The combined effects of Mlp1/2 and Nup2 create a dynamic network of interactions that regulate the spatial organization of NPCs within the nucleus.
Other molecular mechanisms involved in mediating the preferential localization of NPCs to the non-nucleolar territory may include the binding of specific nuclear factors or signaling molecules that regulate NPC movement and anchoring. Further research is needed to fully elucidate the intricate network of interactions that govern NPC distribution in the nucleus.

Can the doRITE and single NPC tracking approach be applied to study NPC organization and dynamics in other eukaryotic systems beyond budding yeast?

The doRITE and single NPC tracking approach developed in budding yeast can be adapted and applied to study NPC organization and dynamics in other eukaryotic systems. The principles of doRITE, which involve recombination-induced tag exchange to track individual NPCs through multiple cell cycles, can be implemented in other model organisms with stable protein complexes like NPCs. By tagging specific nucleoporins with fluorescent markers and inducing recombination, researchers can visualize and track the movement of individual NPCs in different cellular contexts.
Single NPC tracking using advanced microscopy techniques can provide valuable insights into the dynamics of NPC assembly, stability, and interactions with other cellular components. This approach can be applied to diverse eukaryotic systems, including mammalian cells, Drosophila, and C. elegans, to investigate the spatial organization and regulation of NPCs in different cellular environments. By combining doRITE with single NPC tracking, researchers can uncover novel mechanisms governing NPC behavior and function across various organisms, enhancing our understanding of nuclear transport and organization.