Search

Fluorescently targeted proteins are widely used for studies of intracellular organelles dynamic. Peripheral proteins are transiently associated with organelles and a significant fraction of them are located at the cytosol. Image analysis of peripheral proteins poses a problem on properly discriminating membrane-associated signal from the cytosolic one. In most cases, signals from organelles are compact in comparison with diffuse signal from cytosol. Commonly used methods for background estimation depend on the assumption that background and foreground signals are separable by spatial frequency filters. However, large non-stained organelles (e.g., nuclei) result in abrupt changes in the cytosol intensity and lead to errors in the background estimation. Such mistakes result in artifacts in the reconstructed foreground signal. We developed a new algorithm that estimates background intensity in fluorescence microscopy images and does not produce artifacts on the borders of nuclei.

APPL1 vesicles represent a distinct population of Rab5-positive early endosomes that undergoes fusion and fission and sorts and exchanges cargo with EEA1 endosomes, suggesting a new organization of the endocytic pathway., Endocytosis allows cargo to enter a series of specialized endosomal compartments, beginning with early endosomes harboring Rab5 and its effector EEA1. There are, however, additional structures labeled by the Rab5 effector APPL1 whose role in endocytic transport remains unclear. It has been proposed that APPL1 vesicles are transport intermediates that convert into EEA1 endosomes. Here, we tested this model by analyzing the ultrastructural morphology, kinetics of cargo transport, and stability of the APPL1 compartment over time. We found that APPL1 resides on a tubulo-vesicular compartment that is capable of sorting cargo for recycling or degradation and that displays long lifetimes, all features typical of early endosomes. Fitting mathematical models to experimental data rules out maturation of APPL1 vesicles into EEA1 endosomes as a primary mechanism for cargo transport. Our data suggest instead that APPL1 endosomes represent a distinct population of Rab5-positive sorting endosomes, thus providing important insights into the compartmental organization of the early endocytic pathway.

The existence of two primary chloride-binding sites was found on the basis of the study of halorhodopsin spectra at different chloride concentrations. SVD analysis of the spectra revealed two chloride-dependent components at low chloride concentration (0.1-10 mM). Global fitting of SVD components found KD values of 0.47 mM and 5.2 mM with unitity Hill coefficients. The second KD coincides with the apparent KD of the photovoltage response of halorhodopsin.

The intracellular localization of proteins to their specific compartments is a rich source of information for the study of biological processes. The colocalization of a protein with an established compartment marker is routinely measured from multi-color fluorescent microscopy images. Unfortunately, the apparent colocalization is a mixture of real and random colocalization. Random colocalization results from the limited resolution of the light microscope and the close location or occlusion of objects in the crowded cytoplasmic environment. Commonly used methods for the correction of random colocalization work well if the random colocalization is significantly smaller than the real colocalization. In the case where they have comparable values, the final result could be a senseless negative value. To solve this problem, we first developed a probabilistic model for the estimation of random colocalization and demonstrated that it produces results that coincide with the standard scramble method. Second, we developed a probabilistic model for the correction of random colocalization for the double and multiple colocalization of intracellular markers on vesicular structures. Our probabilistic method of estimation of real colocalization has two main advantages: 1) it never gives a negative colocalization value and 2) provides the estimation of colocalization uncertainty.

The scheme of the bacteriorhodopsin photocycle associated with a transmembrane proton transfer and electrogenesis is considered. The role of conformational changes in the polypeptide chain during the proton transport is discussed.

The flash-induced voltage response of halorhodopsin at high NaCl concentration comprises two main kinetic components. The first component with tau approximately 1 micros does not exceed 4% of the overall response amplitude and is probably associated with the formation of the L (hR520) intermediate. The second main component with tau approximately 1-2.5 ms which is independent of Cl- concentration can be ascribed to the transmembrane Cl- translocation during the L intermediate decay. The photoelectric response in the absence of Cl- has the opposite polarity and does not exceed 6% of the overall response amplitude at high NaCl concentration. A pH decrease results in substitution of the Cl(-)-dependent components by the photoresponse which is similar to that in the absence of Cl-. Thus, the difference between photoresponses of chloride-binding and chloride-free halorhodopsin forms resembles that of bacteriorhodopsin purple neutral and blue acid forms, respectively. The photovoltage data obtained can hardly be explained within the framework of the photocycle scheme suggested by Varo et al. [Biochemistry 34 (1995), 14490-14499]. We suppose that the O-type intermediate belongs to some form of halorhodopsin incapable of Cl- transport.

Retromer, a peripheral membrane protein complex, plays an instrumental role in host of cellular processes by its ability to recycle receptors from endosomes to the trans-Golgi network. It consists of two distinct sub-complexes, a membrane recognizing, sorting nexins (SNX) complex and a cargo recognition, vacuolar protein sorting (Vps) complex. Small GTPase, Rab7 is known to recruit retromer on endosomal membrane via interactions with the Vps sub-complex. The molecular mechanism underlying the recruitment process including the role of individual Vps proteins is yet to be deciphered. In this study, we developed a FRET-based assay in HeLa cells that demonstrated the interaction of Rab7 with Vps35 and Vps26 in vivo. Furthermore, we showed that Rab7 recruits retromer to late endosomes via direct interactions with N-terminal conserved regions in Vps35. However, the single point mutation, which disrupts the interaction between Vps35 and Vps26, perturbed the Rab7-mediated recruitment of retromer in HeLa cells. Using biophysical measurements, we demonstrate that the association of Vps26 with Vps35 resulted in high affinity binding between the Vps sub-complex and the activated Rab7 suggesting for a possible allosteric role of Vps26. Thus, this study provides molecular insights into the essential role of Vps26 and Vps35 in Rab7-mediated recruitment of the core retromer complex.

Laser flash-induced photovoltage responses of the D85S and D85T mutants as well as of the wild-type acid blue form are similar and reflect intraprotein charge redistribution caused by retinal isomerization. The Cl−-induced transition of all of these blue forms into purple ones is accompanied by the appearance of electrogenic stages, which is probably associated with Cl− translocation in the cytoplasmic direction. Cl− translocation efficiency of these purple forms is much lower than that of the proton transport by the wild-type bacteriorhodopsin. The values of the efficiency do not exceed 15, 8 and 3% for the D85T, D85S and wild-type acid purple form, respectively. Cl− induces an additional electrogenic phase in the photovoltage responses of the D85S mutant and the wild-type acid purple form. This phase is supposed to be associated with the reversible Cl− movement in the extracellular direction. It is interesting that this component is absent in the photovoltage response of the D85T mutant which has, like halorhodopsin, a threonine residue at position 85.

Originally the maximum entropy method for exponent deconvolution was restricted to the positive exponent’s amplitudes by the entropy S(f, m) definition. It limits application of the method, since many experimental kinetics show both the rise and the decay, which manifest themselves as positive and negative amplitudes in the exponent spectrum. The generalization of entropy formulation for non‐negative distribution (S. F. Gull and J. Skilling) overcomes this limitation. The drawback of the approach was, that m lost the meaning of the prior distribution, since that maximum of generalized S(f, m) is independent on m and achieved at f ≡ 0. It is significant problem when there are apriori information about possible spectrum behaviour. In the present work some assumptions of the entropy generalization was relaxed and alternative entropy formulation, with non‐uniform prior was used for analysis of simulated and experimental data. The new approach was applied to spectra analysis of the absorption kinetics of the bacteriorhodopsin (bR—light driven proton pump from archea Halobacterium salinarium) photocycle. It was shown that the process of the intermediate M formation is non‐exponential in the wild type bR. The non‐exponential process could be interpreted as result of the protein conformational changes during proton transfer from the Shiff‐base of bR.

In the bacteriorhodopsin-containing proteoliposomes, a laser flash is found to induce formation of a bathointermediate decaying in several seconds, the difference spectrum being similar to the purple–blue transition. Different pH buffers do not affect the intermediate, whereas an uncoupler, gramicidin A, and lipophilic ions accelerate decay of the intermediate or inhibit its formation. In the liposomes containing E204Q bacteriorhodopsin mutant, formation of the intermediate is suppressed. In the wild-type bacteriorhodopsin liposomes, the bathointermediate formation is pH-independent within the pH 5–7 range. The efficiency of the long-lived O intermediate formation increases at a low pH. In the wild-type as well as in the E204Q mutant purple membrane, the O intermediate decay is slowed down at slightly higher pH values than that of the purple–blue transition. It is suggested that the membrane potential affects the equilibrium between the bacteriorhodopsin ground state (Glu-204 is protonated and Asp-85 is deprotonated) and the O intermediate (Asp-85 is protonated and Glu-204 is deprotonated), stabilizing the latter by changing the relative affinity of Asp-85 and Glu-204 to H+. At a low pH, protonation of a proton-releasing group (possibly Glu-194) in the bacteriorhodopsin ground state seems to prevent deprotonation of the Glu-204 during the photocycle. Thus, all protonatable residues of the outward proton pathway should be protonated in the O intermediate. Under such conditions, membrane potential stabilization of the O intermediate in the liposomes can be attributed to the direct effect of the potential on the pK value of Asp-85.

At high ionic strength, the pH dependence of the M intermediate decay in a photocycle of the D96N mutant bacteriorhodopsin shows a complicated behavior which is found to be due to the coexistence of two pathways of the M conversion. The M decay which dominates at pH 5. This pathway is insensitive to glutaraldehyde and some other similar inhibitors (lutetium ions, sucrose and glycerol). On the other hand, it is sensitive to the pK changes of the group X (Glu-204) in the outward proton pathway. Possibly, the M decay through this pathway represents a reverse H+ transport process (the proton uptake from the external surface) and proceeds via the L intermediate.

Summary Pharmacological modulators of host-microbial interactions can in principle be identified using high-content screens. However, a severe limitation of this approach is the lack of insights into the mode of action of compounds selected during the primary screen. To overcome this problem, we developed a combined experimental and computational approach. We designed a quantitative multiparametric image-based assay to measure intracellular mycobacteria in primary human macrophages, screened a chemical library containing FDA-approved drugs, and validated three compounds for intracellular killing of M. tuberculosis . By integrating the multiparametric profiles of the chemicals with those of siRNAs from a genome-wide survey on endocytosis, we predicted and experimentally verified that two compounds modulate autophagy, whereas the third accelerates endosomal progression. Our findings demonstrate the value of integrating small molecules and genetic screens for identifying cellular mechanisms modulated by chemicals. Furthermore, selective pharmacological modulation of host trafficking pathways can be applied to intracellular pathogens beyond mycobacteria.

Electrogenic events in the E204Q bacteriorhodopsin mutant have been studied. A two-fold decrease in the magnitude of microsecond photovoltage generation coupled to M intermediate formation in the E204Q mutant is shown. This means that deprotonation of E204 is an electrogenic process and its electrogenicity is comparable to that of the proton transfer from the Schiff base to D85. pH dependence of the electrogenicity of M intermediate formation in the wild-type bacteriorhodopsin reveals only one component corresponding to the protonation of D85 in the bacteriorhodopsin ground state and transition of the purple neutral form into the blue acid form. Thus, the pK of E204 in the M state is close to the pK of D85 in the bacteriorhodopsin ground state (3) and far below the pK of the terminal proton release group (approximately 6). It is concluded that E204 functions as the intermediate proton donor rather than the terminal proton release group in the bacteriorhodopsin proton pump.