Plasma Membrane Of HeLa Cells Research Articles

AKTs do not translocate to the nucleus upon stimulation but AKT3 can constitutively signal from the nuclear envelope.

AKT is a central signaling protein kinase that plays a role in the regulation of cellular survival metabolism and cell growth, as well as in pathologies such as diabetes and cancer. Human AKT consists of three isoforms (AKT1-3) that may fulfill different functions. Here, we report that distinct subcellular localization of the isoforms directly influences their activity and function. AKT1 is localized primarily in the cytoplasm, AKT2 in the nucleus, and AKT3 in the nucleus or nuclear envelope. None of the isoforms actively translocates into the nucleus upon stimulation. Interestingly, AKT3 at the nuclear envelope is constitutively phosphorylated, enabling a constant phosphorylation of TSC2 at this location. Knockdown of AKT3 induces moderate attenuation of cell proliferation of breast cancer cells. We suggest that in addition to the stimulation-induced activation of the lysosomal/cytoplasmic AKT1-TSC2 pathway, a subpopulation of TSC2 is constitutively inactivated by AKT3 at the nuclear envelope of transformed cells.

Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P2 within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P2 confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PHPLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P2 is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymerization, compartmentalization of PI(4,5)P2 is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P2 domains in HeLa cells.

Engineering and development of model lipid membranes mimicking the HeLa cell membrane

Cells are complex systems whose interaction with nanocarriers, i.e., liposomes, are continuously under investigation to improve drug uptake. Model membranes can facilitate the understanding of the processes involved in fusion or endocytosis. In this work, we engineered two different lipid model membranes, vesicles and supported lipid bilayers (SLBs), mimicking the lipid composition of the HeLa cell plasma membrane. We characterized the model using atomic force microscopy (AFM) and fluorescence. We found that liposomes formed with four lipid components mimicking the HeLa cell bilayer show a liquid ordered fluid nature between 13 °C and 34 °C and yield featureless SLBs onto mica. We evaluated the fusion between the model and liposomes positively charged with and without cholesterol by AFM-based force spectroscopy and fluorescence techniques, such as Förster resonance energy transfer, fluorescence lifetime decay and fluorescence anisotropy. The results indicated a primary electrostatic interaction between the HeLa bilayer model and the liposomes. It was also confirmed the well-known fact that cholesterol enhances the fusion process with the engineered HeLa bilayer. All results support the usefulness of the engineered model in the rationale design of liposomes for drug delivery.

Volumetric Semantic Instance Segmentation of the Plasma Membrane of HeLa Cells

In this work, an unsupervised volumetric semantic instance segmentation of the plasma membrane of HeLa cells as observed with serial block face scanning electron microscopy is described. The resin background of the images was segmented at different slices of a 3D stack of 518 slices with 8192 × 8192 pixels each. The background was used to create a distance map, which helped identify and rank the cells by their size at each slice. The centroids of the cells detected at different slices were linked to identify them as a single cell that spanned a number of slices. A subset of these cells, i.e., the largest ones and those not close to the edges were selected for further processing. The selected cells were then automatically cropped to smaller regions of interest of 2000 × 2000 × 300 voxels that were treated as cell instances. Then, for each of these volumes, the nucleus was segmented, and the cell was separated from any neighbouring cells through a series of traditional image processing steps that followed the plasma membrane. The segmentation process was repeated for all the regions of interest previously selected. For one cell for which the ground truth was available, the algorithm provided excellent results in Accuracy (AC) and the Jaccard similarity Index (JI): nucleus: JI , AC , cell including nucleus JI , AC , cell excluding nucleus JI , AC . A limitation of the algorithm for the plasma membrane segmentation was the presence of background. In samples with tightly packed cells, this may not be available. When tested for these conditions, the segmentation of the nuclear envelope was still possible. All the code and data were released openly through GitHub, Zenodo and EMPIAR.

Nuclear Stiffness Decreases with Disruption of the Extracellular Matrix in Living Tissues.

Reciprocal interactions between the cell nucleus and the extracellular matrix lead to macroscale tissue phenotype changes. However, little is known about how the extracellular matrix environment affects gene expression and cellular phenotype in the native tissue environment. Here, it is hypothesized that enzymatic disruption of the tissue matrix results in a softer tissue, affecting the stiffness of embedded cell and nuclear structures. The aim is to directly measure nuclear mechanics without perturbing the native tissue structure to better understand nuclear interplay with the cell and tissue microenvironments. To accomplish this, an atomic force microscopy needle-tip probe technique that probes nuclear stiffness in cultured cells to measure the nuclear envelope and cell membrane stiffness within native tissue is expanded. This technique is validated by imaging needle penetration and subsequent repair of the plasma and nuclear membranes of HeLa cells stably expressing the membrane repair protein CHMP4B-GFP. In the native tissue environment ex vivo, it is found that while enzymatic degradation of viable cartilage tissues with collagenase 3 (MMP-13) and aggrecanase-1 (ADAMTS-4) decreased tissue matrix stiffness, cell and nuclear membrane stiffness is also decreased. Finally, the capability for cell and nucleus elastography using the AFM needle-tip technique is demonstrated. These results demonstrate disruption of the native tissue environment that propagates to the plasma membrane and interior nuclear envelope structures of viable cells.

Dibenzothiophene Sulfone Derivatives as Plasma Membrane Dyes.

Dibenzothiophene 5,5-dioxide (DBTOO) derivatives have recently been shown to processes utility as fluorescent cell dyes. In an effort to extend the functionality of DBTOO-based dyes to include the visualization of cellular membranes, two lipophilic DBTOO were synthesized and their ability to incorporate into the plasma membrane of HeLa cells was examined by fluorescent microscopy. The photophysical properties of the two new DBTOO derivatives were determined and both have good fluorescent quantum yields and a visible blue emission. Due to agreeable wavelengths of excitation and emission, a standard 4',6-diamindino-2-phenylindole (DAPI) filter set worked well with these dyes. After co-staining, it was confirmed that both DBTOO dyes localized in the plasma membrane. The quality of the overlap was quantified using Pearson correlation coefficient, which indicated a strong overlap between the DBTOO dyes and the standard plasma membrane dye. The novel dyes also displayed relatively low toxicity to the HeLa cells with IC50 between 10 and 100µm. Thus, this work reports a new use of DBTOO derivatives as fluorescent microscopy stains.

Mitotic Catastrophe Induced in HeLa Tumor Cells by Photodynamic Therapy with Methyl-aminolevulinate.

Photodynamic therapy (PDT) constitutes a cancer treatment modality based on the administration of a photosensitizer, which accumulates in tumor cells. The subsequent irradiation of the tumoral area triggers the formation of reactive oxygen species responsible for cancer cell death. One of the compounds approved in clinical practice is methyl-aminolevulinate (MAL), a protoporphyrin IX (PpIX) precursor intermediate of heme synthesis. We have identified the mitotic catastrophe (MC) process after MAL-PDT in HeLa human carcinoma cells. The fluorescence microscopy revealed that PpIX was located mainly at plasma membrane and lysosomes of HeLa cells, although some fluorescence was also detected at endoplasmic reticulum and Golgi apparatus. Cell blockage at metaphase-anaphase transition was observed 24 h after PDT by phase contrast microscopy and flow cytometry. Mitotic apparatus components evaluation by immunofluorescence and Western blot indicated: multipolar spindles and disorganized chromosomes in the equatorial plate accompanied with dispersion of centromeres and alterations in aurora kinase proteins. The mitotic blockage induced by MAL-PDT resembled that induced by two compounds used in chemotherapy, taxol and nocodazole, both targeting microtubules. The alterations in tumoral cells provided evidence of MC induced by MAL-PDT, resolving mainly by apoptosis, directly or through the formation of multinucleate cells.

Modification of cellular membranes conveys cryoprotection to cells during rapid, non-equilibrium cryopreservation.

Rapid cooling and re-warming has been shown promising to cryopreserve living cells, which cannot be preserved by conventional slow freezing methods. However, success is limited by the cytotoxicity of highly concentrated cryoprotective agents. Recent results have shown that cryoprotective agents do not need to suppress intracellular ice crystals completely to allow for survival after cryopreservation. Cryoprotective agents like DMSO or ethylene glycol can also lead to a tolerance of cells towards intracellular ice. It is however unclear by which mechanism this tolerance is achieved. These substances are also known to modulate properties of cellular membranes. It is shown here that cryoprotective DMSO and ethylene glycol have a clear influence on the mobility of lipids in the plasma membrane of HeLa cells. To isolate changes of the properties of plasma membranes from effects on ice formation, the membrane properties were modulated in absence of cryoprotective agents. This was achieved by changing their sterol content. In cells with elevated sterol content, an immobile lipid fraction was present, similar to cells treated with DMSO and ethylene glycol. These cells showed also significantly increased plasma membrane integrity after rapid freezing and thawing in the absence of classical cryoprotective agents. However, their intracellular lysosomes, which cannot be enriched with sterols, still got ruptured. These results clearly indicate that a modulation of membrane properties can convey cryoprotection. Upon slow cooling, elevated sterol content had actually an adverse effect on the plasma membranes, which shows that this effect is specific for rapid, non-equilibrium cooling processes. Unraveling this alternative mode of action of cryoprotection should help in the directed design of novel cryoprotective agents, which might be less cytotoxic than classical, empirically-found cryoprotective agents.

C24 Sphingolipids Govern the Transbilayer Asymmetry of Cholesterol and Lateral Organization of Model and Live-Cell Plasma Membranes

Mammalian sphingolipids, primarily with C24 or C16 acyl chains, reside in the outer leaflet of the plasma membrane. Curiously, little is known how C24 sphingolipids impact cholesterol and membrane microdomains. Here, we present evidence that C24 sphingomyelin, when placed in the outer leaflet, suppresses microdomains in giant unilamellar vesicles and also suppresses submicron domains in the plasma membrane of HeLa cells. Free energy calculations suggested that cholesterol has a preference for the inner leaflet if C24 sphingomyelin is in the outer leaflet. We indeed observe that cholesterol enriches in the inner leaflet (80%) if C24 sphingomyelin is in the outer leaflet. Similarly, cholesterol primarily resides in the cytoplasmic leaflet (80%) in the plasma membrane of human erythrocytes where C24 sphingolipids are naturally abundant in the outer leaflet. We conclude that C24 sphingomyelin uniquely interacts with cholesterol and regulates the lateral organization in asymmetric membranes, potentially by generating cholesterol asymmetry.

A rare missense mutation in GJB3 (Cx31G45E) is associated with a unique cellular phenotype resulting in necrotic cell death

Erythrokeratodermia variabilis et progressiva (EKV-P) is caused by mutations in either the GJB3 (Cx31) or GJB4 genes (Cx30.3). We identified a rare GJB3 missense mutation, c.134G>A (p.G45E), in two unrelated patients and investigated its cellular characteristics. Expression of Cx31G45E-GFP caused previously undescribed changes within HeLa cells and HaCaT cells, a model human keratinocyte cell line. Cx31WT-GFP localised to the plasma membrane, but expression of Cx31G45E-GFP caused vacuolar expansion of the endoplasmic reticulum (ER), the mutant protein accumulated within the ER membrane and disassembly of the microtubular network occurred. No ER stress responses were evoked. Cx31WT-myc-myc-6xHis and Cx31G45E-GFP co-immunoprecipitated, indicative of heteromeric interaction, but co-expression with Cx31WT-mCherry, Cx26 or Cx30.3 did not mitigate the phenotype. Cx31 and Cx31G45E both co-immunoprecipitated with Cx43, indicating the ability to form heteromeric connexons. WT-Cx31 and Cx43 assembled into large gap junction plaques at points of cell-to-cell contact; Cx31G45E restricted the ability of Cx43 to reach the plasma membrane in both HaCaT cells and HeLa cells stably expressing Cx43 where the proteins strongly co-localised with the vacolourised ER. Cell viability assays identified an increase in cell death in cells expressing Cx31G45E-GFP, which FACS analysis determined was necrotic. Blocking connexin channel function with 18α-glycyrrhetinic acid did not completely rescue necrosis or prevent propidium iodide uptake, suggesting that expression of Cx31G45E-GFP damages the cellular membrane independent of its channel function. Our data suggest that entrapment of Cx43 and necrotic cell death in the epidermis could underlie the EKV skin phenotype.

The C‐terminal Tail of TCL Localizes the GTPase to the Plasma Membrane of HeLa Cells

TCL/RhoJ belongs to the Rho family of GTPases, and it has been shown to contribute to angiogenesis and tumor growth. To better understand the inherent biochemistry of TCL, our lab has been investigating primary sequence difference between TCL and its closest homologs Cdc42 and TC10. Thus far, amino acids 17–20 in the N terminus have been found to be important for GTP‐loading and localization of TCL to the plasma membrane, and mutation of this sequence leads to TCL localization to vesicles. To better understand how the TCL N‐terminus, C‐terminus, and GTP‐binding regions contribute to membrane and vesicle localization independently of each other, expression constructs were generated that fuse the first 24 and last 21 amino acids of TCL to the fluorescent protein Venus. Interestingly, these constructs indicate that the C‐terminus alone may be mostly responsible for TCL plasma membrane localization. TCL constructs containing the GTPase core without the C‐terminal tail suggest the GTPase region uniquely controls localization to vesicles. Additional mutations in the C‐terminal tail will be generated to test how the C‐terminal CAAX sequence and the polybasic sequence contribute to TCL localization in order to understand how TCL localization regulates its function.

Mammalian Nonmuscle Myosin II Binds to Anionic Phospholipids with Concomitant Dissociation of the Regulatory Light Chain

Mammalian cells express three Class II nonmuscle myosins (NM): NM2A, NM2B, and NM2C. The three NM2s have well established essential roles in cell motility, adhesion, and cytokinesis and less well defined roles in vesicle transport and other processes that would require association of NM2s with cell membranes. Previous evidence for the mechanism of NM2-membrane association includes direct interaction of NM2s with membrane lipids and indirect interaction by association of NM2s with membrane-bound F-actin or peripheral membrane proteins. Direct binding of NM2s to phosphatidylserine-liposomes, but not to phosphatidylcholine-liposomes, has been reported, but the molecular basis of the interaction between NM2s and acidic phospholipids has not been previously investigated. We now show that filamentous, full-length NM2A, NM2B, and NM2C and monomeric, non-filamentous heavy meromyosin bind to liposomes containing one or more acidic phospholipids (phosphatidylserine, phosphatidylinositol 4,5-diphosphate, and phosphatidylinositol 3,4,5-triphosphate) but do not bind to 100% phosphatidylcholine-liposomes. Binding of NM2s to acidic liposomes occurs predominantly through interaction of the liposomes with the regulatory light chain (RLC) binding site in the myosin heavy chain with concomitant dissociation of the RLC. Phosphorylation of myosin-bound RLC by myosin light chain kinase substantially inhibits binding to liposomes of both filamentous NM2 and non-filamentous heavy meromyosin; the addition of excess unbound RLC, but not excess unbound essential light chain, competes with liposome binding. Consistent with the in vitro data, we show that endogenous and expressed NM2A associates with the plasma membrane of HeLa cells and fibrosarcoma cells independently of F-actin.

The Salmonella effector SteA binds phosphatidylinositol 4-phosphate for subcellular targeting within host cells.

Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4-phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella-containing vacuole (SCV) and to Salmonella-induced tubules; using the PI(4)P-binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N-terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells.

Nanoparticles-cell association predicted by protein corona fingerprints.

In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a "protein corona" layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø≈ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells.

Site of fluorescent label modifies interaction of melittin with live cells and model membranes

The mechanism of membrane disruption by melittin (MLT) of giant unilamellar vesicles (GUVs) and live cells was studied using fluorescence microscopy and two fluorescent synthetic analogues of MLT. The N-terminus of one of these was acylated with thiopropionic acid to enable labeling with maleimido-AlexaFluor 430 to study the interaction of MLT with live cells. It was compared with a second analogue labeled at P14C. The results indicated that the fluorescent peptides adhered to the membrane bilayer of phosphatidylcholine GUVs and inserted into the plasma membrane of HeLa cells. Fluorescence and light microscopy revealed changes in cell morphology after exposure to MLT peptides and showed bleb formation in the plasma membrane of HeLa cells. However, the membrane disruptive effect was dependent upon the location of the fluorescent label on the peptide and was greater when MLT was labeled at the N-terminus. Proline at position 14 appeared to be important for antimicrobial activity, hemolysis and cytotoxicity, but not essential for cell membrane disruption.

Discreet and distinct clustering of five model membrane proteins revealed by single molecule localization microscopy.

Compartmentalization is a functionally important property of the plasma membrane, yet the underlying principles that organize membrane proteins into distinct domains are not well understood. Using single molecule localization microscopy, we assessed the clustering of five model membrane proteins in the plasma membrane of HeLa cells. All five proteins formed discrete and distinct nano-scaled clusters. The extent of clustering of the five proteins, independent of their membrane anchors, increased significantly when the fluorescent protein mEOS2 was employed, suggesting that protein-protein interactions are a key driver for clustering. Further, actin depolymerization or reduction of membrane order had a greater, and in some instances opposing effects on the clustering of membrane proteins fused to mEOS2 compared to PS-CFP2-fusion proteins. The data propose that protein interactions can override the lateral organization imposed by membrane anchors to provide an exquisite regulation of the mosaic-like compartmentalization of the plasma membrane.

The connexin 30.3 of zebrafish homologue of human connexin 26 may play similar role in the inner ear

The intercellular gap junction channels formed by connexins (CXs) are important for recycling potassium ions in the inner ear. CXs are encoded by a family of the CX gene, such as GJB2, and the mechanism leading to mutant connexin-associated diseases, including hearing loss, remains to be elucidated. In this study, using bioinformatics, we found that two zebrafish cx genes, cx27.5 and cx30.3, are likely homologous to human and mouse GJB2. During embryogenesis, zebrafish cx27.5 was rarely expressed at 1.5–3 h post-fertilization (hpf), but a relatively high level of cx27.5 expression was detected from 6 to 96 hpf. However, zebrafish cx30.3 transcripts were hardly detected until 9 hpf. The temporal experiment was conducted in whole larvae. Both cx27.5 and cx30.3 transcripts were revealed significantly in the inner ear by reverse transcription polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization (WISH). In the HeLa cell model, we found that zebrafish Cx27.5 was distributed intracellularly in the cytoplasm, whereas Cx30.3 was localized in the plasma membrane of HeLa cells stably expressing Cx proteins. The expression pattern of zebrafish Cx30.3 in HeLa cells was more similar to that of cells expressing human CX26 than Cx27.5. In addition, we found that Cx30.3 was localized in the cell membrane of hair cells within the inner ear by immunohistochemistry (IHC), suggesting that zebrafish cx30.3 might play an essential role in the development of the inner ear, in the same manner as human GJB2. We then performed morpholino knockdown studies in zebrafish embryos to elucidate the physiological functions of Cx30.3. The zebrafish cx30.3 morphants exhibited wild-type-like and heart edema phenotypes with smaller inner ears at 72 hpf. Based on these results, we suggest that the zebrafish Cx30.3 and mammalian CX26 may play alike roles in the inner ear. Thus, zebrafish can potentially serve as a model for studying hearing loss disorders that result from human CX26 mutations.

Investigating the role of F-actin in human immunodeficiency virus assembly by live-cell microscopy.

Human immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane, which is lined by a dense network of filamentous actin (F-actin). Large amounts of actin have been detected in HIV-1 virions, proposed to be incorporated by interactions with the nucleocapsid domain of the viral polyprotein Gag. Previous studies addressing the role of F-actin in HIV-1 particle formation using F-actin-interfering drugs did not yield consistent results. Filamentous structures pointing toward nascent HIV-1 budding sites, detected by cryo-electron tomography and atomic force microscopy, prompted us to revisit the role of F-actin in HIV-1 assembly by live-cell microscopy. HeLa cells coexpressing HIV-1 carrying fluorescently labeled Gag and a labeled F-actin-binding peptide were imaged by live-cell total internal reflection fluorescence microscopy (TIR-FM). Computational analysis of image series did not reveal characteristic patterns of F-actin in the vicinity of viral budding sites. Furthermore, no transient recruitment of F-actin during bud formation was detected by monitoring fluorescence intensity changes at nascent HIV-1 assembly sites. The chosen approach allowed us to measure the effect of F-actin-interfering drugs on the assembly of individual virions in parallel with monitoring changes in the F-actin network of the respective cell. Treatment of cells with latrunculin did not affect the efficiency and dynamics of Gag assembly under conditions resulting in the disruption of F-actin filaments. Normal assembly rates were also observed upon transient stabilization of F-actin by short-term treatment with jasplakinolide. Taken together, these findings indicate that actin filament dynamics are dispensable for HIV-1 Gag assembly at the plasma membrane of HeLa cells. Importance: HIV-1 particles assemble at the plasma membrane of virus-producing cells. This membrane is lined by a dense network of actin filaments that might either present a physical obstacle to the formation of virus particles or generate force promoting the assembly process. Drug-mediated interference with the actin cytoskeleton showed different results for the formation of retroviral particles in different studies, likely due to general effects on the cell upon prolonged drug treatment. Here, we characterized the effect of actin-interfering compounds on the HIV-1 assembly process by direct observation of virus formation in live cells, which allowed us to measure assembly rate constants directly upon drug addition. Virus assembly proceeded with normal rates when actin filaments were either disrupted or stabilized. Taken together with the absence of characteristic actin filament patterns at viral budding sites in our analyses, this indicates that the actin network is dispensable for HIV-1 assembly.

Development of a FRET Biosensor with High Specificity for Akt

The serine/threonine kinase Akt plays a critical role in cell proliferation, survival, and tumorigenesis. As a central kinase in the phosphatidylinositol 3-kinase pathway, its activation mechanism at the plasma membrane has been well characterized. However, the subcellular Akt activity in living cells is still largely unknown. Fluorescence resonance energy transfer (FRET)-based biosensors have emerged as indispensable tools to visualize the subcellular activities of signaling molecules. In this study, we developed a highly specific FRET biosensor for Akt based on the Eevee backbone, called Eevee-iAkt. Using inhibitors targeting kinases upstream and downstream of Akt, we showed that Eevee-iAkt specifically monitors Akt activity in living cells. To visualize Akt activity at different subcellular compartments, we targeted Eevee-iAkt to raft and non-raft regions of the plasma membrane, mitochondria, and nucleus in HeLa and Cos7 cells. Interestingly, we revealed substantial differences in Akt activation between HeLa and Cos7 cells upon epidermal growth factor (EGF) stimulation: Akt was transiently activated in HeLa cells with comparable levels at the plasma membrane, cytosol, and mitochondria. In contrast, sustained and spatially localized Akt activation was observed in EGF-stimulated Cos7 cells. We found high Akt activity at the plasma membrane, low activity in the cytosol, and no detectable activity at the mitochondria and nucleus in Cos7 cells. The Eevee-iAkt biosensor was shown to be a valuable tool to study the functional relationship between subcellular Akt activation and its anti-apoptotic role in living cells.

A Resealed-Cell System for Analyzing Pathogenic Intracellular Events: Perturbation of Endocytic Pathways under Diabetic Conditions

Cell-based assay systems that can serve as cellular models of aberrant function in pathogenic organs would be novel and useful tools for screening drugs and clarifying the molecular mechanisms of various diseases. We constructed model cells that replicated the conditions in diabetic hepatocytes by using the cell resealing technique, which enables the exchange of cytosol. The plasma membrane of HeLa cells was permeabilized with the streptococcal toxin streptolysin O, and cytosol that had been prepared from wild-type or db/db diabetic mice was introduced into the resulting semi-intact cells. By resealing the plasma membrane by exposure to Ca2+, we created WT or Db model cells, in which the cytosolic conditions replicated those of healthy or diabetic liver. Interestingly, phosphorylation of p38 MAPK was promoted, whereas the level of endosomal phosphatidylinositol-3-phosphate was decreased, in Db cells. We investigated several endocytic pathways in WT and Db cells, and found that retrograde endosome-to-Golgi transport was delayed in a p38 MAPK-dependent manner in Db cells. Furthermore, the degradation pathway of the EGF receptor from endosomes to lysosomes was enhanced in Db cells, and this did not depend on the activation of p38 MAPK. The disease model cell system should become a powerful tool for the detection of aberrant processes in cells under pathogenic conditions and for therapeutic applications.