Quantification of protein delivery in live cells using fluorescence correlation spectroscopy

Knox, Susan L; Steinauer, Angela; Alpha-Cobb, Garrett; Trexler, Adam J.; Rhoades, Elizabeth; Schepartz, Alanna

doi:10.1016/bs.mie.2020.05.007

Cited by 14 publications

(25 citation statements)

References 76 publications

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“…In the following, cell internalization of these peptides is discussed. Several review articles are available dealing with CPPs and fluorescence, we cite here only selected recent papers (Boisquérin et al ., 2015; Uusna et al ., 2015; Barba-Bon et al ., 2019; Seisel et al ., 2019; Deprey and Kritzer, 2020; Knox et al ., 2020; Sakamoto et al ., 2020).…”

Section: Biophysical Methods and Cppsmentioning

confidence: 99%

Studies of cell-penetrating peptides by biophysical methods

Zorko

Langel

2022

Quart. Rev. Biophys.

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Biophysical studies have a very high impact on the understanding of internalization, molecular mechanisms, interactions, and localization of CPPs and CPP/cargo conjugates in live cells or in vivo. Biophysical studies are often first carried out in test-tube set-ups or in vitro, leading to the complicated in vivo systems. This review describes recent studies of CPP internalization, mechanisms, and localization. The multiple methods in these studies reveal different novel and important aspects and define the rules for CPP mechanisms, hopefully leading to their improved applicability to novel and safe therapies.

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Section: Biophysical Methods and Cppsmentioning

confidence: 99%

Studies of cell-penetrating peptides by biophysical methods

Zorko

Langel

2022

Quart. Rev. Biophys.

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“…The autocorrelation data ( Supplementary Figs. 1-3 ) was fitted to a 3D anomalous diffusion equation (cytosol) 42 or a two-component 3D diffusion equation (nucleus) 44 using a custom MATLAB script to establish the concentration of each protein in the cytosol ( c ) and nucleus ( d ) of Saos-2 cells or CHO-K1 cells. Center line, median; box limits, 25-75 percentile; whiskers, 10-90 percentile.…”

Section: Resultsmentioning

confidence: 99%

“…1-3 ). FCS is a single-molecule technique that deconvolutes the time-dependent change in fluorescence in a small cytosolic or nuclear volume to establish both average intracellular concentration as well as diffusion time 42,43 . FCS analysis of Saos-2 cells revealed that all t MeCP2-Rho conjugates localize more significantly to the nucleus than the cytosol ( Extended Data Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The protein secondary structure was predicted (Supplementary Table 1) using the webserver BeStSel (http://bestsel.elte.hu/index.php). -3) from FCS measurements was fitted to a 3D anomalous diffusion equation (cytosol) 42 or a two-component 3D diffusion equation (nucleus) 44 to establish the concentration of each protein in the cytosol and nucleus. Center line, median; box limits, 25-75 percentile; whiskers, 10-90 percentile.…”

Section: Data Availabilitymentioning

confidence: 99%

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Dose-dependent nuclear delivery and transcriptional repression with a cell-penetrant MeCP2

Zhang

Zoltek

Mozumdar

et al. 2022

Preprint

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Methyl-CpG-binding-protein 2 (MeCP2) is a nuclear protein expressed in all cell types, especially neurons. Mutations in the MECP2 gene cause Rett syndrome (RTT), an incurable neurological disorder that disproportionately affects young girls. Strategies to restore MeCP2 expression phenotypically reverse RTT-like symptoms in male and female MeCP2-deficient mice, suggesting that direct nuclear delivery of functional MeCP2 could restore MeCP2 activity. We report that ZF-tMeCP2, a conjugate of MeCP2(∆aa13-71, 313-484) and the cell-permeant mini-protein ZF5.3, both binds DNA in a methylation-dependent manner and reaches the nucleus of model cell lines intact at concentrations above 700 nM. When delivered to live cells, ZF-tMeCP2 engages the NCoR/SMRT co-repressor complex and selectively represses transcription from methylated promoters. Efficient nuclear delivery of ZF-tMeCP2 relies on a unique endosomal escape portal provided by HOPS-dependent endosomal fusion. The Tat conjugate of MeCP2 (Tat-tMeCP2), evaluated for comparison, is degraded within the nucleus, is not selective for methylated promoters, and trafficks in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of protein-derived therapeutics.

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“…In this review, we will focus on the fluorescence-based singlemolecule techniques. Among which, total internal reflection fluorescence (TIRF) microscopy, fluorescence spectroscopy techniques, fluorescence recovery after photobleaching (FRAP) [19], fluorescence resonance energy transfer (FRET) [20], fluorescence correlation spectroscopy (FCS) [21], are especially suitable for cellular signal transduction studies. We will briefly summarize the basic principles, short-comings, and major biological applications of SMD methods for the quantitative investigation of biological signal transduction mechanisms.…”

Section: Introductionmentioning

confidence: 99%

Signal Transduction Mechanisms Quantitatively Observed One Molecule at a Time

Chen

et al. 2022

Front. Phys.

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Improved single-molecule methods can largely increase our understanding of underlying molecular mechanism during cellular signal transduction. In contrast to conventional bulk methods, monitoring molecules one at a time can circumvent averaging effects and acquire unique information. With single-molecule techniques, quantitative characterizations can be achieved at microscopic level, especially for biochemical systems with strong heterogeneity. Here we review four fundamental single-molecule techniques including total internal reflection fluorescence imaging, single-molecule fluorescence recovery after photobleaching, single-molecule Förster resonance energy transfer, and fluorescence correlation/cross-correlation spectroscopy. These techniques are frequently employed in quantitatively investigating the molecular translocation, protein-protein interactions, aggregations, and conformational dynamics involved in the signal transduction both in vitro and in vivo. We also summarized the basic principles and implementations of these single-molecule techniques, as well as the conjunct applications extending the single-molecule measurements to multiple dimensions.

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Quantification of protein delivery in live cells using fluorescence correlation spectroscopy

Cited by 14 publications

References 76 publications

Studies of cell-penetrating peptides by biophysical methods

Studies of cell-penetrating peptides by biophysical methods

Dose-dependent nuclear delivery and transcriptional repression with a cell-penetrant MeCP2

Signal Transduction Mechanisms Quantitatively Observed One Molecule at a Time

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