Fluorescent Recovery after Photobleaching (FRAP) Analysis of Nuclear Export Rates Identifies Intrinsic Features of Nucleocytoplasmic Transport

Cardarelli, Francesco; Tosti, Luca; Serresi, Michela; Beltram, Fabio; Bizzarri, Ranieri

doi:10.1074/jbc.m111.304899

Cited by 20 publications

(22 citation statements)

References 29 publications

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“…Based on the obtained results the authors could argue for a molecular transport process with no significant interference between the nuclear import and export processes, in analogy with the FRAP-based results discussed above [53]. This evidence can be explained, in principle, by assuming that the pores are alternatively involved in export or import processes or that indeed two (structurally?)…”

Section: Single Molecules Through Single Pores: Localization-based Apsupporting

confidence: 62%

Time-resolved biophysical approaches to nucleocytoplasmic transport

Cardarelli

2017

Computational and Structural Biotechnology Journal

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Molecules are continuously shuttling across the nuclear envelope barrier that separates the nucleus from the cytoplasm. Instead of being just a barrier to diffusion, the nuclear envelope is rather a complex filter that provides eukaryotes with an elaborate spatiotemporal regulation of fundamental molecular processes, such as gene expression and protein translation. Given the highly dynamic nature of nucleocytoplasmic transport, during the past few decades large efforts were devoted to the development and application of time resolved, fluorescence-based, biophysical methods to capture the details of molecular motion across the nuclear envelope. These methods are here divided into three major classes, according to the differences in the way they report on the molecular process of nucleocytoplasmic transport. In detail, the first class encompasses those methods based on the perturbation of the fluorescence signal, also known as ensemble-averaging methods, which average the behavior of many molecules (across many pores). The second class comprises those methods based on the localization of single fluorescently-labelled molecules and tracking of their position in space and time, potentially across single pores. Finally, the third class encompasses methods based on the statistical analysis of spontaneous fluorescence fluctuations out of the equilibrium or stationary state of the system. In this case, the behavior of single molecules is probed in presence of many similarly-labelled molecules, without dwelling on any of them. Here these three classes, with their respective pros and cons as well as their main applications to nucleocytoplasmic shuttling will be briefly reviewed and discussed.

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Section: Single Molecules Through Single Pores: Localization-based Apsupporting

confidence: 62%

Time-resolved biophysical approaches to nucleocytoplasmic transport

Cardarelli

2017

Computational and Structural Biotechnology Journal

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“…Nuclear influx and efflux of Foxo occur via two parallel and independent unidirectional transport processes that transport Foxo in opposite directions through the nuclear pores ( Fig. 2) (1,9,20). Only unphosphorylated Foxo is carried into the nuclei by the nuclear import system, and only phosphorylated Foxo is carried out of the nuclei by the nuclear export system (3, 23, 30; see above).…”

Section: Theory: Kinetic Models For Foxo Nuclear Fluxesmentioning

confidence: 99%

Mathematical modeling reveals modulation of both nuclear influx and efflux of Foxo1 by the IGF-I/PI3K/Akt pathway in skeletal muscle fibers

Wimmer

Schachter

Stonko

et al. 2014

American Journal of Physiology-Cell Physiology

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Wimmer RJ, Liu Y, Schachter TN, Stonko DP, Peercy BE, Schneider MF. Mathematical modeling reveals modulation of both nuclear influx and efflux of Foxo1 by the IGF-I/PI3K/Akt pathway in skeletal muscle fibers. Am J Physiol Cell Physiol 306: C570-C584, 2014. First published January 22, 2014; doi:10.1152/ajpcell.00338.2013.-Foxo family transcription factors contribute to muscle atrophy by promoting transcription of the ubiquitin ligases muscle-specific RING finger protein and muscle atrophy F-box/atrogin-1. Foxo transcriptional effectiveness is largely determined by its nuclear-cytoplasmic distribution, with unphosphorylated Foxo1 transported into nuclei and phosphorylated Foxo1 transported out of nuclei. We expressed the fluorescent fusion protein Foxo1-green fluorescent protein (GFP) in cultured adult mouse flexor digitorum brevis muscle fibers and tracked the time course of the nuclear-to-cytoplasmic Foxo1-GFP mean pixel fluorescence ratio (N/C) in living fibers by confocal imaging. We previously showed that IGF-I, which activates the Foxo kinase Akt/PKB, caused a rapid marked decline in N/C, whereas inhibition of Akt caused a modest increase in N/C. Here we develop a two-state mathematical model for Foxo1 nuclear-cytoplasmic redistribution, where Foxo phosphorylation/dephosphorylation is assumed to be fast compared with nuclear influx and efflux. Cytoplasmic Foxo1-GFP mean pixel fluorescence is constant due to the much larger cytoplasmic than nuclear volume. Analysis of N/C time courses reveals that IGF-I strongly increased unidirectional nuclear efflux, indicating similarly increased fractional phosphorylation of Foxo1 within nuclei, and decreased unidirectional nuclear influx, indicating increased cytoplasmic fractional phosphorylation of Foxo1. Inhibition of Akt increased Foxo1 unidirectional nuclear influx, consistent with block of Foxo1 cytoplasmic phosphorylation, but did not decrease Foxo1 unidirectional nuclear efflux, indicating that Akt may not be involved in Foxo1 nuclear efflux under control conditions. New media change experiments show that cultured fibers release IGF-I-like factors, which maintain low nuclear Foxo1 in the medium. This study demonstrates the power of quantitative modeling of observed nuclear fluxes.Akt1; Foxo1; IGF-I; skeletal muscle TRANSCRIPTION OF A GIVEN GENE is controlled by numerous regulatory proteins, including transcription factors, which are primary activators of transcription, as well as numerous other activators, coactivators, repressors, and corepressors (12, 15a). These factors combine to form macromolecular transcriptional regulatory complexes assembled on the promoter regions of the gene in question (16). To participate in such regulatory complexes, the regulatory molecules must be resident in the nucleus. Thus an important aspect of transcriptional regulation is control of the nuclear-cytoplasmic distribution of these regulatory molecules, and the mechanisms controlling nuclearcytoplasmic movements of transcriptional regulatory molecules can thus be important dete...

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“…2B). We performed a diffusion test in which normally trafficked proteins diffuse poorly into a photo-bleached region, while non-trafficked proteins rapidly diffuse into the bleached region (Cardarelli et al 2012). As hypothesized, the normally trafficked WT-LHB diffused poorly while the LHB-Lys40del mutant rapidly diffused to the bleached Heterodimerization of LHB with CGA and LHCGR activation.…”

Section: Signal Transductionmentioning

confidence: 99%

A vital region for human glycoprotein hormone trafficking revealed by an LHB mutation

Potorac

Rivero-Müller

Trehan

et al. 2016

Journal of Endocrinology

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Glycoprotein hormones are complex hormonally active macromolecules. Luteinizing hormone (LH) is essential for the postnatal development and maturation of the male gonad. Inactivating Luteinizing hormone beta (LHB) gene mutations are exceptionally rare and lead to hypogonadism that is particularly severe in males. We describe a family with selective LH deficiency and hypogonadism in two brothers. DNA sequencing of LHB was performed and the effects of genetic variants on hormone function and secretion were characterized by mutagenesis studies, confocal microscopy and functional assays. A 20-year-old male from a consanguineous family had pubertal delay, hypogonadism and undetectable LH. A homozygous c.118_120del (p.Lys40del) mutation was identified in the patient and his brother, who subsequently had the same phenotype. Treatment with hCG led to pubertal development, increased circulating testosterone and spermatogenesis. Experiments in HeLa cells revealed that the mutant LH is retained intracellularly and showed diffuse cytoplasmic distribution. The mutated LHB heterodimerizes with the common alpha-subunit and can activate its receptor. Deletion of flanking glutamic acid residues at positions 39 and 41 impair LH to a similar extent as deletion of Lys40. This region is functionally important across all heterodimeric glycoprotein hormones, because deletion of the corresponding residues in hCG, follicle-stimulating hormone and thyroid-stimulating hormone beta-subunits also led to intracellular hormone retention. This novel LHB mutation results in hypogonadism due to intracellular sequestration of the hormone and reveals a discrete region in the protein that is crucial for normal secretion of all human glycoprotein hormones.

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Fluorescent Recovery after Photobleaching (FRAP) Analysis of Nuclear Export Rates Identifies Intrinsic Features of Nucleocytoplasmic Transport

Cited by 20 publications

References 29 publications

Time-resolved biophysical approaches to nucleocytoplasmic transport

Time-resolved biophysical approaches to nucleocytoplasmic transport

Mathematical modeling reveals modulation of both nuclear influx and efflux of Foxo1 by the IGF-I/PI3K/Akt pathway in skeletal muscle fibers

A vital region for human glycoprotein hormone trafficking revealed by an LHB mutation

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