Analysis of Arc/Arg3.1 Oligomerization In Vitro and in Living Cells.

Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation, long-term depression, and synaptic scaling. Although its mechanisms of action in these forms of synaptic plasticity are not completely well established, the activities of Arc include the remodeling of the actin cytoskeleton, the facilitation of AMPA receptor (AMPAR) endocytosis, and the regulation of the transcription of AMPAR subunits. In addition, Arc has sequence and structural similarity to retroviral Gag proteins and self-associates into virus-like particles that encapsulate mRNA and perhaps other cargo for intercellular transport. Each of these activities is likely to be influenced by Arc's reversible self-association into multiple oligomeric species. Here, we used mass photometry to show that Arc exists predominantly as monomers, dimers, and trimers at approximately 20 nM concentration in vitro. Fluorescence fluctuation spectroscopy revealed that Arc is almost exclusively present as low-order (monomer to tetramer) oligomers in the cytoplasm of living cells, over a 200 nM to 5 µM concentration range. We also confirmed that an α-helical segment in the N-terminal domain contains essential determinants of Arc's self-association.

Statistical analysis of the autocorrelation function in fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a powerful method to measure concentration, mobility, and stoichiometry in solution and in living cells, but quantitative analysis of FCS data remains challenging due to the correlated noise in the autocorrelation function (ACF) of FCS. We demonstrate here that least-squares fitting of the conventional ACF is incompatible with the χ2 goodness-of-fit test and systematically underestimates the true fit parameter uncertainty. To overcome this challenge, a simple method to fit the ACF is introduced that allows proper calculation of goodness-of-fit statistics and that provides more tightly constrained parameter estimates than the conventional least-squares fitting method, achieving the theoretical minimum uncertainty. Because this method requires significantly more data than the standard method, we further introduce an approximate method that requires fewer data. We demonstrate both these new methods using experiments and simulations of diffusion. Finally, we apply our method to FCS data of the peripheral membrane protein HRas, which has a slow-diffusing membrane-bound population and a fast-diffusing cytoplasmic population. Despite the order-of-magnitude difference of the diffusion times, conventional FCS fails to reliably resolve the two species, whereas the new method identifies the correct model and provides robust estimates of the fit parameters for both species.

Significance of endolymphatic sac surgery with and/or without simultaneous cochlea implant surgery in respect of vertigo control and speech perception in patients with Menière's disease.

PURPOSE: The focus on treating patients with Menière's Disease (MD) lies on the reduction of vertigo attacks and the preservation of sensory function. Endolympathic hydrops is considered as an epiphenomenon in MD, which can potentially be altered by endolymphatic sac surgery (ESS). Purpose of the study was to investigate the influences on vertigo control through manipulation of the perilymphatic system with or without ESS. METHODS: Retrospective data analysis of 86 consecutive patients with MD according to current diagnostic criteria after endolymphatic sac surgery alone (ESSalone; n = 45), cochlear implantation (CI) alone (CIalone; n = 12), and ESS with CI (ESS + CI; n = 29), treated at a tertiary referral center. MAIN OUTCOME MEASURES: vertigo control, speech perception pre- and postoperatively. RESULTS: Gender, side, and preoperative treatment were similar in all groups. Age was younger in the ESSalone-group with 56.2 ± 13.0 years (CIalone = 64.2 ± 11.4 years; ESS + CI = 63.1 ± 9.7 years). Definitive MD was present in all the CIalone, in 79.3% of the ESS + CI and in 59.6% of the ESSalone-patients. Likewise, vertigo control rate was 100% in the CIalone, 89.7% in the ESS + CI and 66.0% in the ESSalone-group. CONCLUSIONS: Vertigo control was improved in all three groups, however, superior in groups treated with CI, potentially contributed by the manipulation of both the endo- and perilymphatic systems. A more systematic characterization of the patients with larger case numbers and documentation of follow up data would be needed to evaluate a clinical effect more properly.

Comparison of speech perception in bimodal cochlear implant patients with respect to the cochlear coverage.

BACKGROUND: The hearing success of patients with bimodal fitting, utilizing both a cochlear implant (CI) and a hearing aid (HA), varies considerably: While some patients benefit from bimodal CI and HA, others do not. OBJECTIVES: This retrospective study aimed to investigate speech perception in bimodally fitted patients and compare it with the cochlear coverage (CC). METHODS: The CC was calculated with the OTOPLAN software, measuring the cochlear duct length on temporal bone CT scans of 39 patients retrospectively. The patients were categorized into two groups: CC ≤ 65% (CC500) and CC > 65% (CC600). Monaural speech intelligibility for monosyllables at a sound pressure level (SPL) of 65 dB in a free-field setting was assessed before and after CI at various time points. The two groups, one with preoperative HA and one with postoperative CI, were compared. Additionally, speech intelligibility was correlated with CC in the entire cohort before CI and at the last available follow-up (last observation time, LOT). RESULTS: Overall, there was no significant difference in speech intelligibility between CC500 and CC600 patients, with both groups demonstrating a consistent improvement after implantation. While CC600 patients tended to exhibit earlier improvement in speech intelligibility, CC500 patients showed a slower initial improvement within the first 3 months but demonstrated a steeper learning curve thereafter. At LOT, the two patient groups converged, with no significant differences in expected speech intelligibility. There was no significant relationship between unimodal/unilateral free-field speech intelligibility and CC. Interestingly, patients with a CC of 70-75% achieved the highest speech intelligibility. CONCLUSION: Despite of the lack of a significant correlation between CC and speech perception, patients appeared to reach their maximum in unimodal/unilateral speech perception primarily at a coverage level of 70-75%. Nevertheless, further investigation is warranted, as CC500 was associated with shorter cochlear duct length, and different types of electrodes were used in both groups.

A Good Practice Guide for Organizing the Scientific Program of International Conferences.

This paper provides a step-by-step guide for organizing the scientific program (OSP) of international conferences. Through informal discussions, a panel of experts organizing international conferences came up with this guide, which includes a flowchart, checklist, and detailed discussions of each step. Subsequently, additional specialists were invited to evaluate this synopsis and provide their input. All of the participants approved the final version after the outline was improved. This guide proposes the following six steps: 1) preparation, 2) recruitment, 3) building the agenda, 4) cross-checking the program, 5) reviewing and finalizing, and 6) in-conference refining. Thirteen items are specified across the six main steps in a detailed checklist. This OSP guide includes a flowchart and a checklist for providing a comprehensive manual for establishing, conducting, and organizing international scientific conferences. Understanding the procedures that are expected to be followed when holding a scientific conference enables the involved parties to organize and assign tasks to one another as well as create a schedule that allows them to finish their work on time. This guide can be used at any kind of scientific conference to describe an organized process, resulting in a professional and distinguished scientific program.

Autocorrelation function of finite-length data in fluorescence correlation spectroscopy.

The experimental autocorrelation function of fluorescence correlation spectroscopy calculated from finite-length data is a biased estimator of the theoretical correlation function. This study presents a new theoretical framework that explicitly accounts for the data length to allow for unbiased analysis of experimental autocorrelation functions. To validate our theory, we applied it to experiments and simulations of diffusion and characterized the accuracy and precision of the resulting parameter estimates. Because measurements in living cells are often affected by instabilities of the fluorescence signal, autocorrelation functions are typically calculated on segmented data to improve their robustness. Our reformulated theory extends the range of usable segment times down to timescales approaching the diffusion time. This flexibility confers unique advantages for live-cell data that contain intensity variations and instabilities. We describe several applications of short segmentation to analyze data contaminated with unwanted fluctuations, drifts, or spikes in the intensity that are not suited for conventional fluorescence correlation analysis. These results demonstrate the potential of our theoretical framework to significantly expand the experimental systems accessible to fluorescence correlation spectroscopy.

Partitioning of ribonucleoprotein complexes from the cellular actin cortex.

The cell cortex plays a crucial role in cell mechanics, signaling, and development. However, little is known about the influence of the cortical meshwork on the spatial distribution of cytoplasmic biomolecules. Here, we describe a fluorescence microscopy method with the capacity to infer the intracellular distribution of labeled biomolecules with subresolution accuracy. Unexpectedly, we find that RNA binding proteins are partially excluded from the cytoplasmic volume adjacent to the plasma membrane that corresponds to the actin cortex. Complementary diffusion measurements of RNA-protein complexes suggest that a rudimentary model based on excluded volume interactions can explain this partitioning effect. Our results suggest the actin cortex meshwork may play a role in regulating the biomolecular content of the volume immediately adjacent to the plasma membrane.

Molecular Determinants of Human T-cell Leukemia Virus Type 1 Gag Targeting to the Plasma Membrane for Assembly.

Assembly of human T-cell leukemia virus type 1 (HTLV-1) particles is initiated by the trafficking of virally encoded Gag polyproteins to the inner leaflet of the plasma membrane (PM). Gag-PM interactions are mediated by the matrix (MA) domain, which contains a myristoyl group (myr) and a basic patch formed by lysine and arginine residues. For many retroviruses, Gag-PM interactions are mediated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]; however, previous studies suggested that HTLV-1 Gag-PM interactions and therefore virus assembly are less dependent on PI(4,5)P2. We have recently shown that PI(4,5)P2 binds directly to HTLV-1 unmyristoylated MA [myr(-)MA] and that myr(-)MA binding to membranes is significantly enhanced by inclusion of phosphatidylserine (PS) and PI(4,5)P2. Herein, we employed structural, biophysical, biochemical, mutagenesis, and cell-based assays to identify residues involved in MA-membrane interactions. Our data revealed that the lysine-rich motif (Lys47, Lys48, and Lys51) constitutes the primary PI(4,5)P2-binding site. Furthermore, we show that arginine residues 3, 7, 14 and 17 located in the unstructured N-terminus are essential for MA binding to membranes containing PS and/or PI(4,5)P2. Substitution of lysine and arginine residues severely attenuated virus-like particle production, but only the lysine residues could be clearly correlated with reduced PM binding. These results support a mechanism by which HTLV-1 Gag targeting to the PM is mediated by a trio engagement of the myr group, Arg-rich and Lys-rich motifs. These findings advance our understanding of a key step in retroviral particle assembly.

Suitable Electrode Choice for Robotic-Assisted Cochlear Implant Surgery: A Systematic Literature Review of Manual Electrode Insertion Adverse Events.

Background and Objective: The cochlear implant (CI) electrode insertion process is a key step in CI surgery. One of the aims of advances in robotic-assisted CI surgery (RACIS) is to realize better cochlear structure preservation and to precisely control insertion. The aim of this literature review is to gain insight into electrode selection for RACIS by acquiring a thorough knowledge of electrode insertion and related complications from classic CI surgery involving a manual electrode insertion process. Methods: A systematic electronic search of the literature was carried out using PubMed, Scopus, Cochrane, and Web of Science to find relevant literature on electrode tip fold over (ETFO), electrode scalar deviation (ESD), and electrode migration (EM) from both pre-shaped and straight electrode types. Results: A total of 82 studies that include 8,603 ears implanted with a CI, i.e., pre-shaped (4,869) and straight electrodes (3,734), were evaluated. The rate of ETFO (25 studies, 2,335 ears), ESD (39 studies, 3,073 ears), and EM (18 studies, 3,195 ears) was determined. An incidence rate (±95% CI) of 5.38% (4.4-6.6%) of ETFO, 28.6% (26.6-30.6%) of ESD, and 0.53% (0.2-1.1%) of EM is associated with pre-shaped electrodes, whereas with straight electrodes it was 0.51% (0.1-1.3%), 11% (9.2-13.0%), and 3.2% (2.5-3.95%), respectively. The differences between the pre-shaped and straight electrode types are highly significant (p < 0.001). Laboratory experiments show evidence that robotic insertions of electrodes are less traumatic than manual insertions. The influence of round window (RW) vs. cochleostomy (Coch) was not assessed. Conclusion: Considering the current electrode designs available and the reported incidence of insertion complications, the use of straight electrodes in RACIS and conventional CI surgery (and manual insertion) appears to be less traumatic to intracochlear structures compared with pre-shaped electrodes. However, EM of straight electrodes should be anticipated. RACIS has the potential to reduce these complications.

PI4P-Dependent Targeting of ATG14 to Mature Autophagosomes.

Degradation of autophagosomal cargo requires the tethering and fusion of autophagosomes with lysosomes that is mediated by the scaffolding protein autophagy related 14 (ATG14). Here, we report that phosphatidylinositol 4-kinase 2A (PI4K2A) generates a pool of phosphatidylinositol 4-phosphate (PI4P) that facilitates the recruitment of ATG14 to mature autophagosomes. We also show that PI4K2A binds to ATG14, suggesting that PI4P may be synthesized in situ in the vicinity of ATG14. Impaired targeting of ATG14 to autophagosomes in PI4K2A-depleted cells is rescued by the introduction of PI4P but not its downstream product phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Thus, PI4P and PI(4,5)P2 have independent functions in late-stage autophagy. These results provide a mechanism to explain prior studies indicating that PI4K2A and its product PI4P are necessary for autophagosome-lysosome fusion.

Gain-of-Function Properties of a Dynamin 2 Mutant Implicated in Charcot-Marie-Tooth Disease.

Mutations in the gene encoding dynamin 2 (DNM2), a GTPase that catalyzes membrane constriction and fission, are associated with two autosomal-dominant motor disorders, Charcot-Marie-Tooth disease (CMT) and centronuclear myopathy (CNM), which affect nerve and muscle, respectively. Many of these mutations affect the pleckstrin homology domain of DNM2, yet there is almost no overlap between the sets of mutations that cause CMT or CNM. A subset of CMT-linked mutations inhibit the interaction of DNM2 with phosphatidylinositol (4,5) bisphosphate, which is essential for DNM2 function in endocytosis. In contrast, CNM-linked mutations inhibit intramolecular interactions that normally suppress dynamin self-assembly and GTPase activation. Hence, CNM-linked DNM2 mutants form abnormally stable polymers and express enhanced assembly-dependent GTPase activation. These distinct effects of CMT and CNM mutations are consistent with current findings that DNM2-dependent CMT and CNM are loss-of-function and gain-of-function diseases, respectively. In this study, we present evidence that at least one CMT-causing DNM2 mutant (ΔDEE; lacking residues 555DEE557) forms polymers that, like the CNM mutants, are resistant to disassembly and display enhanced GTPase activation. We further show that the ΔDEE mutant undergoes 2-3-fold higher levels of tyrosine phosphorylation than wild-type DNM2. These results suggest that molecular mechanisms underlying the absence of pathogenic overlap between DNM2-dependent CMT and CNM should be re-examined.

Effect of Heat Exposure on Activity Degradation of Enzymes in Mango Varieties Sindri, SB Chaunsa, and Tommy Atkins during Drying.

Mango has been described as a valuable source of nutrients and enzymes that are beneficial to human health. Drying at different temperatures not only affects the nutritional properties but can also contribute to the degradation of valuable enzymes in dried fruit. The novelty of this paper is to investigate the quality of hot air dried mango in terms of activity retention of the heat-sensitive enzymes (HSE). For this, HSE was first screened in fresh mango flesh of the variety Samar Bahisht (SB) Chaunsa. Later, the combined effect of different drying temperatures (40 °C, 50 °C, 60 °C, 70 °C, and 80 °C) and air velocities (1.0 ms-1 and 1.4 ms-1) on the activity retention of HSE in dried mango slices of the varieties Sindri, SB Chaunsa, and Tommy Atkins were investigated. The results showed that the drying temperature had a significant impact on the degradation of HSE, while at the same time some influence of the air velocity was also observed. Drying at 40 °C and an air velocity of 1.4 ms-1 retained more HSE compared to those samples dried at higher temperatures. The least retention of HSE was found in samples dried at 80 °C.

Time-shifted mean-segmented Q data of a luminal protein measured at the nuclear envelope by fluorescence fluctuation microscopy.

Fluorescence fluctuation microscopy is a widely used method to determine the mobility and oligomeric state of proteins in the live cell environment. Existing analysis methods rely on statistical evaluation of data segments with the implicit assumption that no significant signal fluctuations occur on the time scale of a data segment. Recent work on extending fluorescence fluctuation methods to the nuclear envelope of living cells identified a slow fluctuation process that is associated with the undulations of the nuclear membranes, which lead to intensity fluctuations due to local volume changes at the nuclear envelope. This environment violates the above-mentioned assumption and is associated with biased evaluation of fluorescence fluctuation data by traditional analysis methods, such as the autocorrelation function. This challenge was overcome by the introduction of the time-shifted mean-segmented Q function, which relies on a sliding scale of data segment lengths. Here, we share experimental fluorescence fluctuation data taken at the nuclear envelope and demonstrate the calculation of the time-shifted mean-segmented Q function from the raw data. The data and analysis should be valuable for researchers interested in fluorescence fluctuation techniques and provides an opportunity to examine the influence of slow fluctuations on existing data analysis methods. The data is related to the research article titled "Protein oligomerization and mobility within the nuclear envelope evaluated by the time-shifted mean-segmented Q factor" [1].

Differentiating Luminal and Membrane-Associated Nuclear Envelope Proteins.

The nuclear envelope (NE) consists of two concentric nuclear membranes separated by the lumen, an ∼40-nm-wide fluid layer. NE proteins are implicated in important cellular processes ranging from gene expression to nuclear positioning. Although recent progress has been achieved in quantifying the assembly states of NE proteins in their native environment with fluorescence fluctuation spectroscopy, these studies raised questions regarding the association of NE proteins with nuclear membranes during the assembly process. Monitoring the interaction of proteins with membranes is important because the binding event is often associated with conformational changes that are critical to cellular signaling pathways. Unfortunately, the close physical proximity of both membranes poses a severe experimental challenge in distinguishing luminal and membrane-associated NE proteins. This study seeks to address this problem by introducing new, to our knowledge, fluorescence-based assays that overcome the restrictions imposed by the NE environment. We found that luminal proteins violate the Stokes-Einstein relation, which eliminates a straightforward use of protein mobility as a marker of membrane association within the NE. However, a surprising anomaly in the temperature-dependent mobility of luminal proteins was observed, which was developed into an assay for distinguishing between soluble and membrane-bound NE proteins. We further introduced a second independent tool for distinguishing both protein populations by harnessing the previously reported undulations of the nuclear membranes. These membrane undulations introduce local volume changes that produce an additional fluorescence fluctuation signal for luminal, but not for membrane-bound, proteins. After testing both methods using simple model systems, we apply the two assays to investigate a previously proposed model of membrane association for the luminal domain of SUN2, a constituent protein of the linker of nucleoskeleton and cytoskeleton complex. Finally, we investigate the effect of C- and N-terminal tagging of the luminal ATPase torsinA on its ability to associate with nuclear membranes.

Identifying Heteroprotein Complexes in the Nuclear Envelope.

The nucleus is delineated by the nuclear envelope (NE), which is a double membrane barrier composed of the inner and outer nuclear membranes as well as a ∼40-nm wide lumen. In addition to its barrier function, the NE acts as a critical signaling node for a variety of cellular processes, which are mediated by protein complexes within this subcellular compartment. Although fluorescence fluctuation spectroscopy is a powerful tool for characterizing protein complexes in living cells, it was recently demonstrated that conventional fluorescence fluctuation spectroscopy methods are not suitable for applications in the NE because of the presence of slow nuclear membrane undulations. We previously addressed this challenge by developing time-shifted mean-segmented Q (tsMSQ) analysis and applied it to successfully characterize protein homo-oligomerization in the NE. However, many NE complexes, such as the linker of the nucleoskeleton and cytoskeleton complex, are formed by heterotypic interactions, which single-color tsMSQ is unable to characterize. Here, we describe the development of dual-color (DC) tsMSQ to analyze NE heteroprotein complexes built from proteins that carry two spectrally distinct fluorescent labels. Experiments performed on model systems demonstrate that DC tsMSQ properly identifies heteroprotein complexes and their stoichiometry in the NE by accounting for spectral cross talk and local volume fluctuations. Finally, we applied DC tsMSQ to study the assembly of the linker of the nucleoskeleton and cytoskeleton complex, a heteroprotein complex composed of Klarsicht/ANC-1/SYNE homology and Sad1/UNC-84 (SUN) proteins, in the NE of living cells. Using DC tsMSQ, we demonstrate the ability of the SUN protein SUN2 and the Klarsicht/ANC-1/SYNE homology protein nesprin-2 to form a heterocomplex in vivo. Our results are consistent with previously published in vitro studies and demonstrate the utility of the DC tsMSQ technique for characterizing NE heteroprotein complexes.

Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence.

Delicate and transitory protein engagement at the plasma membrane (PM) is crucial to a broad range of cellular functions, including cell motility, signal transduction, and virus replication. Here, we describe a dual-color (DC) extension of the fluorescence z-scan technique, which has proven successful for quantification of peripheral membrane protein binding to the PM in living cells. We demonstrate that the coexpression of a second, distinctly colored fluorescent protein provides a soluble reference species that delineates the extent of the cell cytoplasm and lowers the detection threshold of z-scan PM-binding measurements by an order of magnitude. DC z-scan generates an intensity profile for each detection channel that contains information on the axial distribution of the peripheral membrane and reference protein. Fit models for DC z-scan are developed and verified using simple model systems. Next, we apply the quantitative DC z-scan technique to investigate the binding of two peripheral membrane protein systems for which previous z-scan studies failed to detect binding: human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein and lipidation-deficient mutants of the fibroblast growth factor receptor substrate 2α. Our findings show that these mutations severely disrupt PM association of fibroblast growth factor receptor substrate 2α but do not eliminate it. We further detected binding of HIV-1 MA to the PM using DC z-scan. Interestingly, our data indicate that HIV-1 MA binds cooperatively to the PM with a dissociation coefficient of Kd ∼16 µM and Hill coefficient of n ∼2.

Quantitative modeling of self-oligomerization of proteins in the nuclear envelope by fluorescence fluctuation analysis.

Analysis of fluorescence fluctuation data through the time-shifted mean-segmented Q (tsMSQ) analysis method has recently been shown to successfully identify protein oligomerization and mobility in the nuclear envelope by properly accounting for local volume fluctuations of the nuclear envelope within living cells. However, by its nature, tsMSQ produces correlated data which poses unique challenges for applying goodness of fit tests and obtaining parameter uncertainties from individual measurements. In this paper, we overcome these challenges by introducing bootstrap tsMSQ which involves randomly resampling the fluorescence intensity data to eliminate the correlations in the tsMSQ data. This analysis technique was verified in both the cytoplasm and the lumen of the nuclear envelope with well-characterized proteins that served as model systems. Uncertainties and goodness of fit tests of individual measurements were compared to estimates obtained from sampling multiple experiments. We further applied bootstrapping to fluorescence fluctuation data of the luminal domain of the SUN domain-containing protein 2 in order to characterize its self-oligomerization within the nuclear envelope. Analysis of the concentration-dependent brightness suggests a monomer-trimer transition of the protein.

Myristoylation-Dependent Palmitoylation of the Receptor Tyrosine Kinase Adaptor FRS2α.

An early step in signaling from activated receptor tyrosine kinases (RTKs) is the recruitment of cytosolic adaptor proteins to autophosphorylated tyrosines in the receptor cytoplasmic domains. Fibroblast growth factor receptor substrate 2α (FRS2α) associates via its phosphotyrosine-binding domain (PTB) to FGF receptors (FGFRs). Upon FGFR activation, FRS2α undergoes phosphorylation on multiple tyrosines, triggering recruitment of the adaptor Grb2 and the tyrosine phosphatase Shp2, resulting in stimulation of PI3K/AKT and MAPK signaling pathways. FRS2α also undergoes N-myristoylation, which was shown to be important for its localization to membranes and its ability to stimulate downstream signaling events (Kouhara et al., 1997). Here we show that FRS2α is also palmitoylated in cells and that cysteines 4 and 5 account for the entire modification. We further show that mutation of those two cysteines interferes with FRS2α localization to the plasma membrane (PM), and we quantify this observation using fluorescence fluctuation spectroscopy approaches. Importantly, prevention of myristoylation by introduction of a G2A mutation also abrogates palmitoylation, raising the possibility that signaling defects previously ascribed to the G2A mutant may actually be due to a failure of that mutant to undergo palmitoylation. Our results demonstrate that FRS2α undergoes coupled myristoylation and palmitoylation. Unlike stable cotranslational modifications, such as myristoylation and prenylation, palmitoylation is reversible due to the relative lability of the thioester linkage. Therefore, palmitoylation may provide a mechanism, in addition to phosphorylation, for dynamic regulation of FRS2 and its downstream signaling pathways.

Protein oligomerization and mobility within the nuclear envelope evaluated by the time-shifted mean-segmented Q factor.

Analysis of fluorescence fluctuation experiments by the mean-segmented Q (MSQ) method was recently used to successfully characterize the oligomeric state and mobility of proteins within the nuclear envelope (NE) of living cells. However, two significant shortcomings of MSQ were recognized. Non-ideal detector behavior due to dead-time and afterpulsing as well as the lack of error analysis currently limit the potential of MSQ. This paper presents time-shifted MSQ (tsMSQ), a new formulation of MSQ that is robust with respect to dead-time and afterpulsing. In addition, a protocol for performing error analysis on tsMSQ data is introduced to assess the quality of fit models and estimate the uncertainties of fit parameters. Together, these developments significantly simplify and improve the analysis of fluorescence fluctuation data taken within the NE. To demonstrate these new developments, tsMSQ was used to characterize the oligomeric state and mobility of the luminal domains of two inner nuclear membrane SUN proteins. The results for the luminal domain of SUN2 obtained through tsMSQ without correction for non-ideal detector effects agree with a recent study that was conducted using the original MSQ formulation. Finally, tsMSQ was applied to characterize the oligomeric state and mobility of the luminal domain of the germline-restricted SUN3.