Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Tom Shani; Moshe Levy; Adrian Israelson

doi:10.3791/55676

Ensayo para medir el transporte nucleocitoplasmático en tiempo real dentro de las células NSC-34 ...

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Neuroscience

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JoVE Journal Neuroscience

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Ensayo para medir el transporte nucleocitoplasmático en tiempo real dentro de las células NSC-34 similares a neuronas motoras

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08:53 min

May 16, 2017

DOI: 10.3791/55676-v

Tom Shani¹, Moshe Levy¹, Adrian Israelson^1,2

¹Department of Physiology and Cell Biology, Faculty of Health Sciences,Ben-Gurion University of the Negev, ²The Zlotowski Center for Neuroscience,Ben-Gurion University of the Negev

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Overview

This protocol describes a sensitive method to measure the nucleocytoplasmic transport rate in motor neuron-like NSC-34 cells. It quantifies real-time changes in the nuclear import of a NLS-NES-GFP protein, aiding in the study of neurogenetic diseases like ALS.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Neurogenetics

Background

Nucleocytoplasmic transport is crucial for cellular function.
ALS and other neurogenetic diseases may disrupt this transport.
Real-time measurement techniques enhance understanding of these processes.
NSC-34 cells serve as a model for studying motor neuron behavior.

Purpose of Study

To detect changes in nucleocytoplasmic transport rates.
To assess the impact of transport rate-suppressive molecules.
To provide insights into neurogenetic diseases like ALS.

Methods Used

Thawing mouse neuroblastoma spinal cord cells.
Centrifugation and resuspension in fresh medium.
Quantification of nuclear import of NLS-NES-GFP protein.
Real-time analysis of transport rates.

Main Results

Demonstrated high sensitivity and quantitative capability.
Real-time analysis revealed minute changes in transport rates.
Identified potential effects of suppressive molecules.
Provided a reliable method for studying nucleocytoplasmic transport.

Conclusions

This method is effective for studying nucleocytoplasmic transport.
It can help elucidate mechanisms underlying neurogenetic diseases.
Future applications may extend to other cell types and conditions.

Frequently Asked Questions

What is nucleocytoplasmic transport?

Nucleocytoplasmic transport is the process by which molecules move between the nucleus and the cytoplasm of a cell.

Why is this transport important?

It is essential for various cellular functions, including gene expression and protein synthesis.

How does ALS affect nucleocytoplasmic transport?

ALS may disrupt the transport process, leading to cellular dysfunction and neurodegeneration.

What are NLS and NES?

NLS (nuclear localization signal) and NES (nuclear export signal) are sequences that direct proteins to enter or exit the nucleus.

What are the advantages of this method?

It is highly sensitive, quantitative, and allows for real-time analysis of transport rates.

Who demonstrated the procedure?

The procedure was demonstrated by Tom Shani, a graduate student from the laboratory.

Este protocolo describe un método para medir sensitivamente la tasa de transporte nucleocitoplasmático dentro de las neuronas motoras NSC-34 células mediante la cuantificación del cambio en tiempo real en la importación nuclear de una proteína NLS-NES-GFP.

El objetivo general de este ensayo es detectar cambios mínimos en la tasa de transporte nucleocitoplasmático de las células en tiempo real en ausencia o presencia de moléculas supresoras de la tasa de transporte potencial. Este método puede ayudar a responder preguntas clave sobre enfermedades neurogenéticas específicas como la ELA, que se ha propuesto que tienen un impacto negativo en el transporte nucleocitoplasmático. Algunas ventajas de esta técnica son que es altamente sensible y cuantitativa y que permite el análisis en tiempo real de cambios mínimos en la tasa de transporte nucleocitoplasmático.

Tom Shani, un estudiante graduado de mi laboratorio, demostrará el procedimiento. Comience descongelando aproximadamente una vez 10 a la sexta células de la médula espinal del neuroblastoma de ratón del almacenamiento de nitrógeno líquido en un baño de agua a 37 grados Celsius. Cuando las células estén completamente descongeladas, recójalas por centrifugación y vuelva a suspender el pellet en un mililitro de medio fresco.

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