Cell Intermediate Filaments Research Articles

Biochemical reactions remote-sensitized by excitons propagating across macroscopic distances along biological fibers: Isomerization of 11-cis-retinal

Presently we report a second example of a biochemical reaction sensitized remotely by excitons (electronic excited states) efficiently transferred along intermediate filaments (IFs) over macroscopic distances. IFs are excellent conductors of energy in the form of excitons, with the efficiency of ca. 0.53 reported in vitro. Excitons were generated by visible light and propagated along Müller cell (MC) intermediate filaments, about 100 μm long. These experiments used a capillary matrix filled by MC IFs extracted from porcine retina. Excitons induced efficient isomerization of 11-cis- to all-trans-retinal, with the reaction quantum yield φcis/trans = 0.347 obtained for 11-cis-retinal concentration of 1.0 × 10−3 M (0.284 g/L) using 546 nm light at 6.87 mW/cm2 power density. Exciton quantum yield φexc at 546 nm was also measured in function of radiation power density, with nonlinearity indicative of biphotonic processes. This is the first case of biphotonic processes occurring at such low light intensities with steady-state illumination. The present results support quantum mechanism of high-contrast vision of vertebrate eyes.

Activation of enzymatic ethanol oxidation by ATP hydrolysis energy transferred to ADH1A enzyme molecule over Müller cell intermediate filaments

Presently exciton activation of enzymatic oxidation of ethanol by human alcohol dehydrogenase (ADH) 1A enzyme is reported. The ADH1A enzyme was activated by infrared (IR) excitons transferred over Müller cell (MC) intermediate filaments (IFs). These IR excitons were generated by energy liberated upon enzymatic ATP hydrolysis and transferred to IFs. Also, the emission spectrum was recorded of the electronically excited ADH1A…NAD+…EtOH complexes obtained by energy transfer from IR excitons that traveled along IFs. These results support the hypothesis that ATP hydrolysis energy may be transmitted in vivo in the form of IR excitons, over the network of IFs, both within and between cells.

IR exciton activation mechanism of ethanol oxidation by human alcohol dehydrogenase (ADH) 1A enzyme

Low-energy (2500 cm−1) exciton transfer was explored in Müller cell (MC) intermediate filaments (IFs) isolated from porcine retina and filling a capillary matrix. Excitons were generated by absorption of IR radiation at 4 μm. The effects of these excitons on ethanol oxidation by human alcohol dehydrogenase (ADH1A) enzyme were quantified. It was found that IR excitons transferred to the enzyme accelerated alcohol oxidation rate, which increased by the factor of 2.76 when exciting the IFs with 2.39 μW/cm2 of 4 μm IR light. Power dependence of the oxidation rate was also explored. These results show that IFs may be transmitting energy to enzyme molecules in vivo, facilitating enzymatic reactions. The required excitons may be produced by cells at the cost of adenosine triphosphate (ATP) hydrolysis energy. Therefore, such control mechanism for enzymatic reactions may be operational in living systems. Direct activation of the enzyme by IR radiation with 4 μm wavelength did not occur; instead, indirect activation of the IF…ADH1A…NAD (nicotinamide adenine dinucleotide)…EtOH complex occurred by energy transfer of the IR exciton to the ADH1A molecule of this complex. Considering that every living cell has a network of IFs, a similar reaction control mechanism may be operational in vivo, providing a much faster energy supply redirection within the cell than ATP diffusion, and justifying a closer inquiry.

Impact of noise on the regulation of intracellular transport of intermediate filaments

Noise affects all biological processes from molecules to cells, organisms and populations. Although the effect of noise on these processes is highly variable, evidence is accumulating which shows natural stochastic fluctuations (noise) can facilitate biological functions. Herein, we investigate the effect of noise on the transport of intermediate filaments in cells by comparing the stochastic and deterministic formalizations of the bidirectional transport of intermediate filaments, long elastic polymers transported along microtubules by antagonistic motor proteins (Dallon et al., 2019; Portet et al., 2019). By numerically exploring discrepancies in timescales and attractors between both formalizations, we characterize the impact of stochastic fluctuations on the individual and ensemble transport. Biologically, we find that noise promotes the collective movement of intermediate filaments and increases the efficiency of its regulation by the biochemical properties of motor-cargo interactions. While stochastic fluctuations reduce the impact of the initial distributions of motor proteins in cells, the number of binding sites and the affinity of motor-cargo interactions are the key parameters controlling transport efficiency and efficacy.

Mitochondrial ATP synthesis activated by exciton energy transfer from Müller cell intermediate filaments

• Exciton energy transferred from IFs to mitochondria accelerated ATP production rate. • ATP production rate explored in function of mitochondrial concentration. • ATP production rate explored in function of intensity of light that produced excitons. ATP production by mitochondria isolated from yeast cells ( S. cerevisiae ) was accelerated by exciton energy transfer from intermediate filaments (IFs). The effect was dependent on mitochondrial concentration and the intensity of light used to generate excitons. Presumably, mitochondrial cytochrome C (CC) was activated by IF exciton energy transfer to CC in the inner mitochondrial membrane (IMM), generating electronically excited CC, and the respective excitation energy was then used in the ATP production chain. A qualitative model was proposed describing observations, although more experiments are required before the detailed mechanisms could be deduced. The present results support the earlier proposed hypothesis of ATP production in vertebrate retina by excess photons in daytime, when retina needs extra energy for recovering the used opsins. Additionally, it shows that at early stages of evolution mitochondria could have been using solar radiation to produce ATP.

Temperature dependence of IR exciton emission spectra in Müller cell intermediate filaments

Temperature dependences of IR exciton properties in Müller cell (MC) intermediate filaments (IFs) isolated from porcine retina were studied. It was found that the widths of the spectral emission bands in the 2500 cm−1 and 5000 cm−1 energy ranges grow with temperature. It was found that temperature effects on the bandwidth may be described by thermal activation of the low-frequency vibrational modes of the IFs. The average activation energies for the two IR bands were estimated. Considering the dynamics of IR emission, its buildup time was independent on the sample temperature, while its decay time decreased with temperature. Thus, the emission decay rate increased exponentially with the sample temperature. The mechanisms explaining the observed temperature effects were proposed and discussed. Taking into account that MC IFs are capable of transmitting ATP hydrolysis energy within and between cells, with these properties being apparently common for all IFs, these IFs may be used by cells for physical energy transport and communications. As presently reported, temperature effects upon IR exciton spectra should not affect these proposed physiological functions to any significant extent. Therefore, the currently reported data are important for improving our understanding of the physical communication mechanisms operating within and between cells.

Effects of pulsed electric fields on exciton propagation efficiency along Müller cell intermediate filaments. Possible separation mechanism of high- and low-contrast images by the eye-brain system

In the current study, we tested a possible mechanism of low- and high-contrast image component discrimination by the vertebrate eye-brain system. Apparently the eye-brain system has to discriminate between the low-contrast image component formed by light scattered within the retina, due to interaction of photons with cells and their parts, and the high-contrast image component transmitted by excitons via the quantum mechanism. Presently, effects of pulsed electric fields applied to Müller cell (MC) intermediate filaments (IFs) on the efficiency of exciton propagation were explored. The effects of both pulse duration and amplitude were recorded. These experimental results show that the eye-brain system may be using signal modulation to discriminate between high- and low-contrast image components, improving our understanding of high-contrast vision in vertebrates.

Energy transfer along Müller cell intermediate filaments isolated from porcine retina: II. Excitons at 2500 cm−1 produced by ADH1A upon hydrolysis of one ATP molecule

Low-energy (IR ~2500 cm−1) exciton transfer was explored in Müller cell (MC) intermediate filaments (IFs) isolated from porcine retina and filling a capillary matrix. IR excitons were generated either by absorption of IR radiation at 4 μm, or by ATP hydrolysis energy transfer to the MC IFs. ATP hydrolysis was catalyzed by human alcohol dehydrogenase (ADH1A) in ADH1A…NAD+…IF complexes. Each exciton was generated by hydrolysis of one ATP molecule. Exciton energy was independent on ATP concentration. The emission spectra of IR excitons arriving to the other side of the capillary matrix were dependent on the exciton generation method. Time-resolved experiments with direct exciton generation by pulsed IR radiation allowed to measure exciton travel velocity, along with the exciton emission time. The results obtained support new ideas on ATP energy transfer along protein filaments in vivo. These unique experimental results provide for detailed understanding of the electronic state structure of the MC IFs. This study reports that MC IFs isolated from porcine retina have a long-living low-lying electronic excited state at 2500 cm−1 above the ground state.

Neural Differentiation of Mouse Embryonic Stem Cells-An in vitro Approach to Profile DNA Methylation of Reprogramming Factor Sox2-SRR2.

Sox2 is one of the core transcription factors maintaining the embryonic stem cells (ES) pluripotency and, also indispensable for cellular reprogramming. However, limited data is available about the DNA methylation of pluripotency genes during lineage-specific differentiations. This study investigated the DNA methylation of Sox2 regulatory region 2 (SRR2) during directed differentiation of mouse ES into neural lineage. ES cells were first grown to form embryoid bodies in suspension which were then dissociated, and cultured in defined medium to promote neural differentiation. Typical neuronal morphology together with the up-regulation of Pax6, neuroepithelial stem cell intermediate filament and β-tubulin III and, down-regulation of pluripotency genes Oct4, Nanog and Sox2 showed the existence of neural phenotype in cells undergoing differentiation. Three CpGs in the core enhancer region of neural-specific SRR2 were individually investigated by direct DNA sequencing post-bisulfite treatment and, found to be unmethylated in differentiated cells at time-points chosen for analysis. This analysis does not limit the possibility of methylation at other CpG sites than those profiled here and/or transient methylation. Hence, similar analyses exploring the DNA methylation at other regions of the Sox2 gene could unravel the onset and transitions of epigenetic signatures influencing the outcome of differentiation pathways and neural development. The data presented here shows that in vitro neural differentiation of embryonic stem cells can be employed to study and characterize molecular regulatory mechanisms governing neurogenesis by applying diverse pharmacological and toxicological agents.

Energy transfer along Müller cell intermediate filaments isolated from porcine retina: I. Excitons produced by ADH1A dimers upon simultaneous hydrolysis of two ATP molecules

IR exciton propagation was explored in Müller cell (MC) intermediate filaments (IFs) filling a capillary matrix. These IFs have been isolated from porcine retina using different methods, while their properties were almost identical. Therefore, IFs isolated from the whole retinas were used presently. IR excitons were generated by IR radiation at 2 μm wavelength, or by enzymatic ATP hydrolysis, with the energy transferred to IFs. Excitons produced by ATP hydrolysis required simultaneous energy contribution of two ATP molecules, indicating simultaneous hydrolysis of two ATP molecules in the naturally dimeric human alcohol dehydrogenase enzyme (ADH1A). ATP hydrolysis was thus catalyzed by ADH1A…NAD+ enzymatic complexes absorbed at the IF extremities protruding out of the capillary matrix. The IR emission spectra of excitons were dependent on the exciton generation method. We believe this resulted from the exciton energy distribution varying in function of the generation method used. The latter seems reasonable, given the very long excited-state lifetimes, implying low nonradiative relaxation rates. The energy liberated by ATP hydrolysis has been measured directly in these experiments, for the first time. The results demonstrate that contrary to the predictions of equilibrium thermodynamics, the liberated energy is independent on the ATP/ADP concentration ratio, indicating that non-equilibrium reactions take place. Time-resolved experiments with excitons produced by pulsed IR radiation evaluated characteristic exciton propagation and emission times. For the first time, biexcitonic processes were observed in biological objects, whereby simultaneous hydrolysis of two ATP molecules bound to the same dimeric ADH1A molecule generated excitons carrying twice the energy liberated by hydrolysis of a single ATP molecule. The results reported indicate that ATP-liberated energy may be transmitted along natural polypeptide nanofibers in vivo, within and between live cells. These ideas could promote new understanding of the biophysics of life.

The Possible Effect of β -carotene on Nicotine Withdrawal in Testicular Tissue of Adult Male Albino Rats. Histological and Immunohistochemical Study.

AbstractBackground and Objectives: Nicotine in cigarette smoking possesses damaging effect on testis. This study was designed to evaluate and compare the prophylactic and therapeutic effects of β carotene on nicotine induced testicular damage during nicotine withdrawal.Materials and Methods:Thirty-nine adult male albino rats were used, 9 rats were control (GI), the remaining rats divided equally into 5 groups: GII (β carotene), GIII (Nicotine), GIV(Nicotine withdrawal), GV (Nicotine withdrawal therapeutic), GVI (Nicotine withdrawal prophylactic group). β carotene (10mg/kg/day) was injected intraperitoneally for 4 weeks. Nicotine (4 mg/kg/day) was injected intraperitoneally for 4 weeks. Before sacrifaction, blood samples were drawn to assess serum levels of malondialdehyde (MDA) and testosterone. After sacrifaction, testicular sections were stained with H&E, Masson trichrome (to reveal collagen fiber deposition and distribution) and immunohistochemically for Vimentin (Sertoli cell intermediate filament(. Morphometric measurements were done by computerized image analysis.Results:Groups I and II showed similar results with normal testicular histological architecture, biochemical and morphometrical results. Deterioration of histological architecture, biochemical and morphometric parameters were recorded in groups III, IV and V (mostly GIII) with significant decrease in diameter of seminiferous tubules, height of spermatogenic epithelium, area percent of vimentin reaction and significant increase in area percent of collagen fibres deposition compared to GI. GVI showed better results than GIII, GIV and GV with restoration of normal testicular histological structure.Conclusion: β carotene could help in improving spermatogenesis. Administration of β carotene prophylactically during nicotine exposure and withdrawal gives better histological, biochemical and morphometrical results than therapeutic administration during nicotine withdrawal period only.

Abstract 5986: Inverse relationship between Giant Axonal Neuropathy gene expression and EMT in human tumors

Abstract Gigaxonin, the protein product of the Giant axonal neuropathy (GAN) gene, is a E3 ubiquitin ligase involved in neural cell and fibroblast intermediate filament processing. We have previously shown that Gigaxonin interacts with p16 in cisplatin-mediated ubiquitination of NF-κB. We analyzed cervical and head and neck cancer cell lines and primary tumors and have found exon 8 c.1293C>T single nucleotide polymorphism (SNP) to occur in both HPV positive and negative tumor cells lines and primary tumors. The prevalence of this SNP in the tumor samples was double that of the normal population (47.25% vs. 22%) and there was no relationship to HPV status. Cell lines containing the SNP showed higher expression of gigaxonin, and an inverse relationship to in vitro tumor cell line growth and cisplatin sensitivity. There was a direct relationship between the expression of gigaxonin and e-cadherin and inverse relationship to the expression of twist, snail and n-cadherin. CRISPR-Cas9 mediated conversion of T to C allele in gigaxonin overexpressing cervical cancer cell line ME 180 resulted in decreased gigaxonin expression accompanied by increased expression of twist, and snail and increased in vitro cell growth. Re-expression of gigaxonin in the CRISPR-Cas9 clone with a plasmid gigaxonin cDNA construct resulted in decreased expression of twist pointing to gigaxonin mediated downregulation of the EMT marker. We therefore hypothesize that GAN, associated with the neurodegenerative disease, is also a human tumor cell growth suppressor gene. Citation Format: Mysore S. Veena, Daniel Sanghoon Shin, Chan Jeong, Jenna R. Chatoff, Natarajan Venkatesan, Saroj K. Basak, David Gray, Sharon P. Wilczynski, Sharon P. Wilczynski, Steven J. Gray, Donald B. Kohn, Marilene B. Wang, Eri S. Srivatsan. Inverse relationship between Giant Axonal Neuropathy gene expression and EMT in human tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5986.

Absorption spectra of Müller cell intermediate filaments: Experimental results and theoretical models.

Experimental spectra of Müller cell (MC) intermediate filaments (IFs) isolated from porcine retina are reported in this work. The absorption spectra recorded at different MC IF concentrations were used to estimate their absorption cross-sections at different wavelengths. The average absorption cross-section of a single MC IF was ca. (0.97 … 2.01)×10-10cm2 in the 650-445nm spectral range. To interpret these experimental absorption spectra, we made ab initio calculations of the optical spectra of α-helix polypeptides, and also used a simplified theoretical approach that modeled an IF by a conductive wire. The energy spectra of the refractive index, extinction coefficient (absorption cross-section), energy loss and reflectivity functions for different photon polarizations, with strong anisotropy with respect to the system axis, were calculated ab initio for polyglycine α-helix molecule containing 1000 glycine residues. Strong anisotropy of these parameters was explained by photons interacting with different electronic transitions. Note that similarly strong anisotropy was also obtained for the optical absorption cross-sections in the simplified model. Both modeling approaches were used for calculating the absorption cross-sections of interest. As a result, the absorption cross-section for photons propagating axially along MC IFs was much larger than their geometrical cross-section. The latter result was explained taking into account the density of electronic states, with numerous electrons contributing to the transition intensity at a given energy. We found that the simple conductive wire model describes the MC IF absorption spectrum better than the ab initio spectra. The latter conclusion was explained by the limitations of ab initio analysis, which only took into account one α-helix with 1000 aminoacids, whereas each porcine Müller cell IF is assembled of thousands of protein molecules, reaching the total length of ca. 100μm. The presently reported results contribute to the understanding of the quantum mechanism of high-contrast vision of vertebrate eyes.

Volt-ampere characteristics of porcine retinal Müller cell intermediate filaments

In the current study we reported current-voltage (I/V) characteristics of Müller cell (MC) intermediate filaments (IFs) isolated from porcine retina. It was found that the measured I/V dependences demonstrate behavior of a semiconductor in contact with metal (Au) electrodes. The analysis of the temperature dependence of the experimental I/V curves produced estimates of the parameter values characterizing the electrical conductivity properties of the studied MC IFs. The I/V characteristics and the parameter values allowed to describe the MC IFs as a semiconducting material. The observed properties clarify the mechanism of high-contrast daylight vision of vertebrate eyes. This mechanism was extensively discussed earlier, and is directly dependent on the electric conductivity properties of MC IFs. Significance StatementRetinal cones and rods are physically connected to glial Müller cells by intermediate filaments (IFs). Electric conductivity of IFs allows for a simple quantum mechanical description of light energy transmission through the retina, providing a convincing mechanism that achieves high-contrast vision in vertebrate eyes. Note that classic light transmission is hindered by light scattering on the retinal structures. Electric conductivity of IFs also opens the possibility for energy exchange within and between other cell types by way of IFs and microtubules, and the possibility that such structures may be used for signaling e.g. in the nervous system.

Energy propagation along polypeptide α-helix: Experimental data and ab initio zone structure

We report new theoretical and experimental results that may significantly change our ideas on the workings of life. We performed ab initio analysis of the band structure of periodic α-helix polypeptides (PP) in function of the chain length. Three different calculation approaches were tested: (a) PP described semiempirically as a one-dimensional object, with the amino acids substituted by effective atoms; (b) density functional theory (DFT) as implemented in WIEN2k approach, and (c) CRYSTAL-17 software package. The approach (c) was used for the detailed ab initio analysis, as it provided better accuracy in less computation time. We found that the bandgap was weakly dependent on the PP composition, with the asymptotic values in the 0.43 – 0.63 eV range. We estimated the effective electron and hole masses, their mean free path and mobility for the glycine-PP. The electron mobility in the PP conductive band was about half of that in polycrystalline silicon. The PP zone structure was used to study the mechanism of energy transfer along the PP. The current-voltage (I/V) characteristics of Müller cell (MC) intermediate filaments (IFs) from porcine retina were experimentally measured. The measured I/V characteristics show that the IFs behave as semiconductors. These results were discussed in light of the presently reported PP zone structure theory. The results obtained may open new areas in biomedical research and applications.

Quantum mechanism of light energy propagation through an avian retina

Taking into account the ultrastructure of the Pied Flycatcher foveal retina reported earlier and the earlier reported properties of Müller cell (MC) intermediate filaments (IFs) isolated from vertebrate retina, we proposed a quantum mechanism (QM) of light energy transfer from the inner limiting membrane level to visual pigments in the photoreceptor cells. This mechanism involves electronic excitation energy transfer in a donor-acceptor system, with the IFs excited by photons acting as energy donors, and visual pigments in the photoreceptor cells acting as energy acceptors. It was shown earlier that IFs with diameter 10 nm and length 117 μm isolated from vertebrate eye retina demonstrate properties of light energy guide, where exciton propagates along such IFs from MC endfeet area to photoreceptor cell area. The energy is mostly transferred via the contact exchange quantum mechanism. Our estimates demonstrate that energy transfer efficiencies in such systems may exceed 80–90%. Thus, the presently developed quantum mechanism of light energy transfer in the inverted retina complements the generally accepted classic optical mechanism and the mechanism whereby Müller cells transmit light like optical fibers. The proposed QM of light energy transfer in the inverted retina explains the high image contrast achieved in photopic conditions by an avian eye, being probably also active in other vertebrates.

New unique optical and electric properties of intermediate filaments in Müller cells

Presently we report new unique optical and electric properties of Müller cell (MC) intermediate filaments (IFs). We inform that these IFs extracted from porcine retina are excellent conductors of light and electric current. Such IF properties may endow vertebrate eyes with high-contrast vision. The properties of the IFs allow a simple quantum-mechanical description that justifies the quantum mechanism (QM) for the light energy transfer between the inner and the outer limiting membranes. These properties also provide direct and unequivocal proof that QM works even in isolated IFs, while the classic theory admits no capacity for light transmission by objects that are so thin. Note that the length and diameter of the IFs were 117 ± 1.3 μm and 10.1 ± 0.07 nm, respectively. The QM avoids the light scattering effects, which could significantly reduce the visual contrast, by conducting light energy in the form of excitons (excited states). This scientific breakthrough may provide new insights for medical ophthalmology.

Optical transparency and electrical conductivity of intermediate filaments in Müller cells and single-wall carbon nanotubes

Presently we investigated the electrical conductivity and optical transparency of Müller cell intermediate filaments. For comparison, the same properties were also explored in the model system of single-wall carbon nanotubes. We report the method of separation and purification of porcine (Sus scrofa domestica) intermediate filaments, extracted from the retinal Müller cells. We also report experimental and theoretical methods of measurements and calculations of the resistivity and light transmission yield by the intermediate filaments and single wall carbon nanotubes. The measured resistivity values were (4.7 ± 0.3) × 10−4 and (2.8 ± 0.2) × 10−4 Ω⋅m−1⋅cm2 at 5 °C (278 K), for the intermediate filaments and single wall carbon nanotubes, respectively, being quite close to those of typical metals. We report a method for measuring the light energy transmission by these nanostructures. We found that they efficiently transfer excitation energy along their axis, with the light reemitted at their other end. The measured yields of transferred light energy were 0.50 ± 0.03 and 0.26 ± 0.02 for intermediate filaments and single wall carbon nanotubes, respectively (λexc = 546.1 nm; T = 288 K). The reported results are novel, providing a direct confirmation of the earlier proposed quantum mechanism of light energy transport in the inverted retina of vertebrates. Our data also show that Müller cell intermediate filaments, in addition to their cytoskeletal function, are capable of providing for the light energy transfer within the inverted retina. The data obtained enable a significant progress in our understanding of the high-contrast vision of the vertebrate eyes. The most important conclusion of the current study is the discovery of light energy propagation along natural biological nanofibers (intermediate filaments). This result is completely novel and unique, being reported for the first time.

Single lipoaminoglycoside promotes efficient intracellular antibody delivery: A comprehensive insight into the mechanism of action

Delivery of biologically active proteins into cells is emerging as important strategy for many applications. Previous experiments have shown that lipoaminoglycosides were capable of delivery of the anti-cytokeratin8 antibody (anti-K8) but only when formulated with lipid helpers potentially leading to toxicity from excess lipids. Here, we optimized anti-K8 delivery with various lipoaminoglycosides in the absence of a lipid helper. Results led to the identification of the aminoglycoside lipid dioleyl phosphoramido ribostamycin (DOPRI) as a potent intracellular delivery system for anti-K8. Electron microscopy revealed that delivered anti-K8 molecules were bound to intermediate filaments in cells. Anti-K8 was bound to the surface of DOPRI vesicles without perturbing lipid organization. Macropinocytosis and caveolin mediated endocytosis contributed to anti-K8 internalization and to filament labeling with a major contribution being made by the caveolin pathway. The results showed that the unique properties of DOPRI were sufficient for efficient intracellular protein delivery without requiring lipid helpers.

Keratin 8/18 regulation of insulin receptor signaling and trafficking in hepatocytes through a concerted phosphoinositide-dependent Akt and Rab5 modulation.

Keratins (Ks) are epithelial cell intermediate filament (IF) proteins that are expressed as pairs in a differentiation-regulated manner. Hepatocyte IFs are made only of K8/K18 pairs, which means that a K8 loss in K8-null mice leads to degradation of K18. Functionally, there is accumulating evidence that IFs contribute to signaling platforms. Here, we investigate the role of K8/K18 IFs in the regulation of insulin receptor (IR) signaling and trafficking in hepatocytes. We find that the IR substrate 1 (IRS1)/PI3K/Akt signaling cascade-downstream of IR-displays prolonged activation in K8-null compared with wild-type hepatocytes. Assessment of the Akt/mammalian target of rapamycin complex 1-mediated feedback loop to IRS1/PI3K, in the absence or presence of drug inhibitors, further supports a preferential K8/K18 IF intervention at the surface membrane. In K8-null hepatocytes, IR trafficking vesicles that are labeled by Rab5/EEA1/phosphatidylinositol 3-phosphate accumulate at a juxtanuclear region via a microtubule-dependent process. Moreover, interference with phosphatidylinositol 4,5-biphosphate signaling aggravates IR/Rab5 accumulation. Overall, results uncover K8/K18 IF regulation of IR signaling via a concerted modulation of phosphatidylinositol 4,5-biphosphate-dependent IRS1/PI3K/Akt signaling and Rab5/phosphatidylinositol 3-phosphate/microtubule trafficking in hepatocytes.-Roux, A., Loranger, A., Lavoie, J. N., Marceau, N. Keratin 8/18 regulation of insulin receptor signaling and trafficking in hepatocytes through a concerted phosphoinositide-dependent Akt and Rab5 modulation.