Fluorescent protein (FP) tagging is widely used in imaging experiments to investigate the subcellular distribution of proteins. However, because the fluorescence of most FP chromophores is quenched upon their protonation, their fluorescence intensities are dependent on their pKas and on the environmental pH. Thus, the concentration of a protein tagged with EGFP (pKa = 6.0) is dramatically underestimated in the lysosomal lumen (pH ~4.7) compared to that of the same protein tagged with mCherry (pKa = 4.5). In this study, we examined the effect of differential FP tagging on the apparent subcellular distribution of several proteins that reside on the cytoplasmic surfaces of secretory/endocytic organelles. Due to the presumed uniformity of cytoplasmic conditions (pH ~7.2–7.4), we expected to find essentially complete overlap of fluorescent signals, regardless of the nature of the fused FP. However, we were surprised to observe significant discrepancies in the apparent distributions of a subset of proteins tagged with EGFP vs. mCherry (Pearson’s correlation coefficients of about 0.80). These discrepancies were not evident when comparing proteins tagged with mCherry vs. other FPs with low pKas (e.g., mTurquoise (pKa = 4.5), mCerulean (pKa = 3.2)) (Pearson’s correlation coefficients of about 0.90–0.95). Our results suggest that FP tags may be sensitive to the microenvironments on the cytoplasmic surfaces of different organelles.

Although near-infrared II (NIR-II) fluorescence imaging (FI) for accurate and noninvasive diagnosis of solid cancer has been developed as one of the crucial methods for formulating appropriate clinical treatment strategies, it is still hindered by the limited availability of NIR-II nanoprobes. In this study, we aim to develop high performance and biocompatible NIR-II probes based on novel mechanism of the aggregation of NIR fluorephores. Using domain effect of liposomal nanostructures, the J-aggregated state of indocyanine (ICG) NIR-II nanoprobes were elaborately fabricated. Combined with molecular dynamics simulation, the molecular mechanism of the formation of J-aggregated state of ICG molecules was discovered. Results show that the engineered J-aggregated state of ICG with a concentration below 50 mu g/mL can fine tune ICG interactions instead of ICG-intermolecular interactions within liposomal structure. The rational designed ICG cationic liposomes (IJA-CLPs) show 3-fold enhancement in NIR-II intensity compared to the conventional ICG liposomes (ICG-CLPs). Furthermore, for a mouse model of mammary tumors with lymph node metastasis, IF7 peptide-modified IJA-CLPs (IF7-IJA-CLPs) can successfully discriminate tumor cell-metastasized lymph nodes with more than 4-fold fluorescence signal improvement. Thus, it is expected to provide a new avenue for designing NIR-II fluorescent probes for enriching the clinical NIR imaging applications.

Gold nanorods (AuNRs), as versatile sensing materials, have wide analytical applications due to their unique optical properties. Cetyltrimethylammonium bromide (C16TAB), a conventional reagent in AuNR synthesis, also often acts as a stabilizer of AuNRs in applications. However, C16TAB-stabilized AuNRs undergo severe spontaneous aggregation and etching under extreme pH conditions, greatly limiting their optical sensing applications. Herein, we accidentally discovered that octadecyl trimethylammonium bromide (C18TAB), a rarely used surfactant for AuNRs, has a substantially higher stabilizing ability than C16TAB in preventing spontaneous aggregation and etching of AuNRs, which enables C18TAB-stabilized AuNRs as a superior sensing platform, demonstrating a 100-fold higher sensitivity than C16TAB-stabilized AuNRs for detection of model analytes. The excellent stability of C18TAB-stabilized AuNRs can be attributed to the higher surfactant coverage density on the gold surface, evidenced by the red-shifted longitudinal band (5 nm), which is tuned by the metal surface refraction index. The experimental results show that C18TAB-stabilized AuNRs can keep monodispersed and unchanged optical properties at very acidic and alkaline conditions with a low concentration of surfactant (0.05 mM). Moreover, the C18TAB-stabilized AuNRs can prevent spontaneous etching in the acidic sensing system and maintain their unchanged plasmon band, therefore decreasing the intensity of the noise signal. Benefiting from these findings, we established a reliable and ultrasensitive C18TAB-stabilized AuNR sensing platform and achieved the ultrasensitive detection of the model biomarker p-aminophenol (pAP), with a visual detection limit of 8 nM. This sensitivity represents at least a 100-fold improvement over the existing method using C16TAB-stabilized AuNRs. Moreover, C18TAB-stabilized AuNRs were successfully applied to detect pAP in urine samples with satisfactory recovery rates of 99.84-114.91%, further validating its reliability in practical applications. In summary, C18TAB-stabilized AuNRs provide a powerful tool for trace-level visual detection in chemo- and biosensing.

β-amyloid (Aβ) aggregates are critical biomarkers for the early diagnosis of Alzheimer's disease (AD). Numerous studies have shown that blood-based detection of Aβ aggregates can achieve similar or higher diagnostic accuracy than clinical cerebrospinal fluid tests. In this study, a reagent-free, anti-fouling electrochemical sensor based on a peptide aptamer was developed. This sensor exhibited anti-fouling and specific recognition capabilities, enabling highly sensitive and selective quantitative detection of Aβ aggregates in blood. The multifunctional peptide (MF-peptide) incorporated a hydrophilic sequence of alternating lysine (K) and glutamic acid (E) residues into the Aβ recognition peptide. The MF-peptide sensor was fabricated via the self-assembly of the MF-peptide onto a working electrode modified with gold nanoparticles. Experimental results revealed that the sensor exhibited excellent sensitivity to Aβ aggregates, with a strong bilinear response over a range of 0.3 fM-0.5 pM and a detection limit of 0.1 fM. The entire detection process was completed within 25 min. Moreover, the sensor displayed strong resistance to high concentrations of endogenous interferents (interference coefficient <12.76 %) and excellent selectivity in the presence of both interferents and Aβ aggregates. Additionally, the sensor was successfully used to accurately detect Aβ aggregates in human serum, maintaining its excellent bilinear response. This study presents a novel, efficient, and rapid strategy for detecting Aβ aggregates in human serum without complex pre-processing and provides practical support for the early clinical diagnosis of AD.

Cuproptosis, a copper-dependent cell death, emerges as a potential anticancer strategy but still faces challenges of systemic toxicity from exogenous copper supplementation, tumor adaptation via glutathione (GSH)-mediated detoxification, and compensatory copper-efflux upregulation. These limitations impede mitochondrial respiratory dysfunction and proteotoxic stress that are essential for cuproptosis, highlighting the demand for tumor-specific copper metabolic modulation. Here, we engineer multifunctional nanoliposomes (DSF/S1P/ISDN-Lipos) that hijack endogenous copper transport for spatially controlled tumor-specific cuproptosis induction while enabling real-time therapeutic monitoring via gas enhanced ultrasonography. Modularly assembled from tumor-targeting sphingosine-1-phosphate (S1P), GSH-responsive nitric oxide (NO) prodrug isosorbide dinitrate (ISDN), and copper-chelator disulfiram (DSF), this DSF/S1P/ISDN-Lipos first facilitates blood-brain tumor barrier traversal and glioblastoma-specific accumulation. Then, intratumorally GSH converts DSF to dithiocarbamate (DTC), chelating endogenous copper into Cu(DTC)2 complexes on the liposome surface. Following internalization, coreleased Cu(DTC)2 and ISDN-derived NO deplete GSH while suppressing ATP7B efflux pumps, amplifying copper overload to trigger lipoylated protein aggregation and Fe-S cluster degradation. Notably, NO-generated ultrasound contrast enables spatiotemporal mapping of copper transport dynamics. In vivo, DSF/S1P/ISDN-Lipos demonstrated favorable biosafety and significantly suppressed orthotopic glioblastoma growth. This work presents a theranostic approach for metal homeostasis regulation, where gas therapy synergizes with endogenous metallo-reprogramming to overcome adaptive resistance.

Boosting the accuracy and speed of cancer detection is highly desirous in tumor detection, and sensors capable of detecting carcinoembryonic antigen (CEA) have great application prospects in this field. A highly sensitive sensor is constructed based on the fluorescence resonance energy transfer (FRET) with heavily rare-earth-doped upconversion nanoparticles (UCNPs) as energy donors and polydopamine nanoparticles (PDA NPs) as energy acceptors. This sensor detects the fluctuations in CEA molecules via luminescence quenching and recovery resulting from a competitive binding assay between CEA and PDA NPs. The high-level-doped design of UCNPs (i.e., NaYF4@NaYbF4:1%Tm@NaYF4) is beneficial, providing upconversion luminescence intensity that is more than 10 times higher than that of the conventional low-level-doped UCNPs (i.e., NaYF4@NaYF4:20%Yb, 0.2%Tm@NaYF4). The sensor exhibits impressive sensitivity. Specifically, in diluted fetal bovine serum, the detection limit reaches 0.013 ng/mL in the range of 0–1.5 ng/mL (S/N = 3), while the detection limit is 1.38 ng/mL in the range of 1.5–250 ng/mL (S/N = 3). This method has great potential for future applications in the rapid and early diagnosis and treatment of cancer.

Compact and user-friendly nucleic acid biosensors play a crucial role in advancing infectious disease research, particularly for coronavirus (COVID-19). While nanophotonic metasurface sensors hold promise for high-performance sensing, they face challenges due to their complexity and bulky readout instruments. In this study, we propose a gradient nanoplasmonic imaging (GNI) metasurface that incorporates the concept of an optical potential well, enabling label-free single-step detection of SARS-CoV-2 sequences. The metasurface sensor consists of nanopillars with continuous variations, forming an optical potential well that results in a centimeter-scale dark ring. This dynamic well exhibits high sensitivity to refractive index changes, recorded by a CCD. To further enhance the visualized sensing performance, plasmonic coupling of gold nanoparticles with the gold nanostructure is employed. Our metasurface-based biosensor achieves rapid single-step detection of SARS-CoV-2 sequences, with a low detection limit of 77.2 pM and a detection range of 0.1-100 nM. This biosensor not only demonstrates exceptional reproducibility and outstanding detection performance, but also maintains remarkable specificity in differentiating SARS-CoV-2 from other diseases with similar symptoms. This simple and spectrometer-free refractometric sensing scheme enables the construction of a compact and cost-efficient prototype. Our imaging-based metasurface biosensing strategy demonstrates valuable merits for rapid, sensitive, and quantitative detection, showcasing its potential as a valuable on-site nucleic acid diagnostic tool.

OBJECTIVE:To explore the effect of shikonin on autophagy and apoptosis of human promyelocytic leukemia cells and its possible mechanism.METHODS:Human promyelocytic leukemia cells NB4 in the logarithmic growth phase were divided into control group (untreated NB4 cells), shikonin group (0.3 µmol/L shikonin treatment), 740Y-P group (15 µmol/L PI3K/Akt/mTOR pathway activator 740Y-P treatment), shikonin+740Y-P group (0.3 µmol/L shikonin and 15 µmol/L 740Y-P co-treatment), after 24 hours of treatment, the cells were used for subsequent experiments. CCK-8 method was used to detect cell viability, monodansylcadaverine (MDC) staining to detect the aggregation of autophagic vesicles, flow cytometry to detect cell apoptosis, and Western blot to detect the expression of Beclin1, LC3, p62, Bax, cleaved caspase-3, Bcl-2 and PI3K/Akt/mTOR pathway related proteins.RESULTS:Compared with the control group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were increased in the shikonin group, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were decreased (all P < 0.05). Compared with the control group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were decreased, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were increased in the 740Y-P group (all P < 0.05). Compared with the shikonin group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were decreased, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were increased in the shikonin+740Y-P group (all P < 0.05). Compared with the control group, the expression of PI3K/Akt/mTOR pathway related proteins p-PI3K, p-Akt, and p-mTOR in NB4 cells were significantly decreased in the shikonin group, while those in the 740Y-P group were increased (all P < 0.05). Compared with the shikonin group, the expressions of p-PI3K, p-Akt, and p-mTOR proteins in NB4 cells were significantly increased in the shikonin+740Y-P group (all P < 0.05).CONCLUSION:Shikonin may promote autophagy and apoptosis of NB4 cells by inhibiting PI3K/Akt/mTOR pathway.

GABARAP is a member of the ATG8 family of ubiquitin-like autophagy related proteins. It was initially discovered as a facilitator of GABA-A receptor translocation to the plasma membrane and has since been shown to promote the intracellular transport of a variety of other proteins under non-autophagic conditions. We and others have shown that GABARAP interacts with the Type II phosphatidylinositol 4-kinase, PI4K2A, and that this interaction is important for autophagosome-lysosome fusion. Here, we identify a 7-amino acid segment within the PI4K2A catalytic domain that contains the GABARAP interaction motif (GIM). This segment resides in an exposed loop that is not conserved in the other mammalian Type II PI 4-kinase, PI4K2B, explaining the specificity of GABARAP binding to the PI4K2A isoform. Mutation of the PI4K2A GIM inhibits GABARAP binding and PI4K2A-mediated recruitment of cytosolic GABARAP to subcellular organelles. We further show that GABARAP binds to mono-phosphorylated phosphoinositides, PI3P, PI4P, and PI5P, raising the possibility that these lipids contribute to the binding energies that drive GABARAP-protein interactions on membranes.

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.

It has long been desired to develop rapid methods for the rapid identification and quantification of pesticides and their metabolites. Carbofuran, a representative pesticide of the carbamate group, is highly systemic and is used on vegetables, fruits and grains, which has led many countries to test for residues in food and the environment. In this study, gold and silver composite core-shell (Au@Ag) nanoparticles were used to label the carbofuran antibody and Raman molecule 5,5-dithiobis-2-nitrobenzoic acid (DTNB) to synthesize Raman immune probes. The signal value of DTNB was read using a Raman spectrometer, and the quantitative detection technology of carbofuran was established based on lateral flow immunochromatographic assay (ICA) combined with surface-enhanced Raman spectroscopy (SERS). SERS-ICA is a rapid, quantitative and ultrasensitive test for the determination of carbofuran in fruits and vegetables with a sensitivity of 0.1 pg mL-1. Consequently, the results demonstrate that the SERS-based lateral flow immunosensor developed in this study has the advantages of excellent assay sensitivity and remarkable multiplexing capability, and thus it will have great application potential in food safety monitoring.

Extracellular vesicles (EVs) have emerged promisingly as natural nanocarriers for drug delivery in the treatment of various diseases due to their low immunogenicity, long circulation time, and ability to cross the blood-brain barrier. However, the low drug loading efficiency seriously hinders the research of EV-based drug delivery. Fusion EVs with liposomes, which have shown powerful drug encapsulation capability, is a novel method to improve the drug loading efficiency for EVs. However, the mechanism of fusion between EVs and liposomes is still unclear. The evaluation of fusion efficiency remains challenging due to the nanoscale size and large heterogeneity of liposomes and EVs, as well as the lack of characterization methods with high sensitivity. By combining Rayleigh scattering and single molecule fluorescence detection in a sheathed flow, our group has developed nano-flow cytometry (nFCM) that is capable of high-sensitivity and high-throughput detection of individual lipid nanomedicines and EVs as small as 40 nm in diameter. In this study, by using FITC-labeled EVs and DiD-labeled liposomes as the model system, membrane fusion between EVs and liposomes was characterized by dual -laser commercial nano-flow cytometer (NanoFCM) through simultaneous light scattering and dual fluorescence detection. The influence of fusion strategies including incubation and freeze & thawing, particle concentration ratio, and surface charge of liposomes were investigated. This study provided important technical support and guidance for the exploration of EV-liposome fusion mechanism and efficient drug delivery based on EVs.

本文通过对全国范围内具有代表性的PC桶企业在原材料控制方面的现场调查数据及相关信息进行分析,了解我国PC桶产品质量及行业发展现状,分析生产过程中原材料控制方面存在的隐患,评价PC桶生产企业对原材料的控制水平及食品安全标准的执行效力.本次PC桶原材料控制过程风险调查,采用现场检查企业原材料控制8项检查条款的方式,结果表明,在26家生产企业中,有147个"符合",占比70.67％;55个"基本符合",占比26.44％,有6个"不符合",占比2.88％,其中8家企业的检查项目全部为"符合",占比30.77％,18家企业存在"基本符合"项,占比69.23％,1家企业存在"不符合"项,占比3.85％.由此可见,我国PC桶生产企业在原材料控制方面仍存在一定风险.

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.

The past decade has become an important strategy in precision medicine for the targeted therapy of many diseases, expecially various types of cancer. As a promising targeted element, nucleic acid aptamers are single-stranded functional oligonucleotides which have specific abilities to bind with various target molecules ranging from small molecules to entire organisms. They are often named 'chemical antibody' and have aroused extensive interest in diverse clinical studies on account of their advantages, such as considerable biostability, versatile chemical modification, low immunogenicity and quick tissue penetration. Thus, aptamer-embedded drug delivery systems offer an unprecedented opportunity in bioanalysis and biomedicine. In this short review, we endeavor to discuss the recent advances in aptamer-based targeted drug delivery platforms for cancer therapy. Some perspectives on the advantages, challenges and opportunities are also presented.

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.

Fluorescence z-scan analysis aims to fit the intensity traces recorded while moving a two-photon excitation volume vertically through a cell in order to identify the vertical concentration profile of a fluorescent species. Z-scan analysis has proved able to quantify delicate PM-protein binding interactions as well as sub-resolution partitioning of proteins due to the actin cortex. Despite recent progress, questions remain regarding the reliability and applicability of z-scan analysis in the complex environment of the living cell. In high precision z-scan applications, obtaining a good quality of fit is critical to ensuring that experimental results remain uncontaminated by fluorescent features outside the scope of the z-scan modeling. However, a statistical method to robustly assess the quality of fit in z-scan analysis has been lacking. To address these issues, we provide control data validating core aspects of the z-scan method at high precision and demonstrate the potential for error when applying the method without rigorous quality of fit controls. We propose a conceptual framework for estimating the amplitude of errors in z-scan analysis due to fluorescent features that may not be included in the z-scan fit model. We apply this framework to analyze the potential for microvilli structures, abundant in some cell lines, to perturb z-scan measurements and we outline data quality controls that contain the potential for error. This work provides a foundation supporting the use of simple stratified layers to model concentration profiles within the living cell together with checks that identify when such simplified models may be inapplicable for a given level of precision. This work has been supported by grants from the National Institutes of Health (R01 GM064589, R01 GM098550, RO1 GM124279).

Severe acute respiratory coronavirus-2 (SARS-CoV-2) is the causative agent of Coronavirus-19 Disease (COVID-19) and remains a severe public health threat. SARS-CoV-2 particles contain four structural proteins - i.e., membrane (M), nucleocapsid (N), spike (S) and envelope (E). Early in virus assembly, the M, S and E proteins are inserted into the rough endoplasmic reticulum (ER). Upon their transport to the ER- Golgi intermediate compartment (ERGIC), there is interaction with N - which initiates particle biogenesis. While fluorescence microscopy offers a non-invasive method to quantitatively study the structural proteins expressed inside living cells, quantitative fluorescence microscopy studies of SARS-CoV-2 assembly have been thus far lacking in the literature. Here, we present work examining the SARS-CoV-2 structural proteins in living cells using fluorescence techniques. We begin by introducing a fluorescence toolkit for our studies which consists of fluorescent protein chimeras of M, S and N. We additionally are developing a virus-like particle (VLP) system and methods of VLP detection to further investigate particle biogenesis. Lastly, we present initial results using quantitative fluorescence microscopy techniques, such as superresolution imaging, fluorescence fluctuation spectroscopy and two-photon scanning, and report preliminary measurements of SARS-CoV-2 structural protein localization, mobility and complex stoichiometry.

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.

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.