Filamentous fungi that colonize microenvironments, such as animal or plant tissue or soil, must find optimal paths through their habitat, but the biological basis for negotiating growth in constrained environments is unknown. We used time-lapse live-cell imaging of Neurospora crassa in microfluidic environments to show how constraining geometries determine the intracellular processes responsible for fungal growth. We found that, if a hypha made contact with obstacles at acute angles, the Spitzenkörper (an assembly of vesicles) moved from the center of the apical dome closer to the obstacle, thus functioning as an internal gyroscope, which preserved the information regarding the initial growth direction. Additionally, the off-axis trajectory of the Spitzenkörper was tracked by microtubules exhibiting "cutting corner" patterns. By contrast, if a hypha made contact with an obstacle at near-orthogonal incidence, the directional memory was lost, due to the temporary collapse of the Spitzenkörper-microtubule system, followed by the formation of two "daughter" hyphae growing in opposite directions along the contour of the obstacle. Finally, a hypha passing a lateral opening in constraining channels continued to grow unperturbed, but a daughter hypha gradually branched into the opening and formed its own Spitzenkörper-microtubule system. These observations suggest that the Spitzenkörper-microtubule system is responsible for efficient space partitioning in microenvironments, but, in its absence during constraint-induced apical splitting and lateral branching, the directional memory is lost, and growth is driven solely by the isotropic turgor pressure. These results further our understanding of fungal growth in microenvironments relevant to environmental, industrial, and medical applications.

Intracellular calcium acts as a secondary messenger in a wide variety of crucial biological signaling processes. Advances in fluorescence microscopy and calcium sensitive dyes has led to the routine quantification of calcium responses in non-excitable cells. However, the automatization of global intracellular calcium analysis at the single-cell level within a large population simultaneously remains challenging. One software, CalQuo (Calcium Quantification), offers some automatic features in calcium analysis. Here, we present an advanced version of the software package: CalQuo 2 . CalQuo 2 analyzes the calcium response in the Fourier-domain, allowing the number of user-defined filtering parameters to be reduced to one and a greater diversity of calcium responses to be recognized, compared to CalQuo that directly interprets the calcium intensity signal. CalQuo 2 differentiates cells that release a single calcium response and those that release oscillatory calcium fluxes. We have demonstrated the use of CalQuo 2 by measuring the calcium response in genetically modified Jurkat T-cells under varying ligand conditions, in which we show that peptide:MHCs and anti-CD3 antibodies trigger a fraction of T cells to release oscillatory calcium fluxes that increase with increasing koff rates. These results show that CalQuo 2 is a robust and user-friendly tool for characterizing global, single cell calcium responses.

During the transition from a healthy state to a cancerous one, cells alter their metabolism to increase proliferation. The underlying metabolic alterations may be caused by a variety of different regulatory events on the transcriptional or post-transcriptional level whose identification contributes to the rational design of therapeutic targets. We present a mechanistic strategy capable of inferring enzymatic regulation from intracellular metabolome measurements that is independent of the actual mechanism of regulation. Here, enzyme activities are expressed by the space of all feasible kinetic constants (k-cone) such that the alteration between two phenotypes is given by their corresponding kinetic spaces. Deriving an expression for the transformation of the healthy to the cancer k-cone we identified putative regulated enzymes between the HeLa and HaCaT cell lines. We show that only a few enzymatic activities change between those two cell lines and that this regulation does not depend on gene transcription but is instead post-transcriptional. Here, we identify phosphofructokinase as the major driver of proliferation in HeLa cells and suggest an optional regulatory program, associated with oxidative stress, that affects the activity of the pentose phosphate pathway.

T7 expression system is an extremely popular approach for the recombinant protein production in Escherichia coli for structural and functional studies and therapeutic applications. There are many useful tools and successful techniques that allow expressing the desired protein in this system. However, high yield of soluble protein often requires a systematic optimization of a wide range of cell cultivation parameters. Here we analyze the effect of three key cultivation parameters - chemical inductor, temperature and time of post-induction culturing on the expression level of TLR1 intracellular TIR domain in a soluble form. In addition, the influence of Triton X-100 detergent on the protein solubility during the cell lysis was investigated. We show that a high expression level of the correctly folded soluble protein can be obtained under different combinations of cultivation parameters.

This work aims to demonstrate the need for in silico design via numerical simulation to produce optimal Fe3O4-based magnetic nanoparticles (MNPs) for magnetic hyperthermia by minimizing the impact of intracellular environments on heating efficiency. By including the relevant magnetic parameters, such as magnetic anisotropy and dipolar interactions, into a numerical model, the heating efficiency of as prepared colloids was preserved in the intracellular environment, providing the largest in vitro specific power absorption (SPA) values yet reported. Dipolar interactions due to intracellular agglomeration, which are included in the simulated SPA, were found to be the main cause of changes in the magnetic relaxation dynamics of MNPs under in vitro conditions. These results pave the way for the magnetism-based design of MNPs that can retain their heating efficiency in vivo, thereby improving the outcome of clinical hyperthermia experiments.

Cytokines are multifunctional small proteins that have a vital influence on inflammatory states of tissues and play a role in signalling and cellular control mechanisms. Cytokine expression has primarily been viewed as a form of direct secretion of molecules through an active transportation; however, other forms of active transport such as extracellular vesicles are at play. This is particularly important in stem cells where signalling molecules are key to communication managing the levels of proliferation, migration, and differentiation into mature cells. This study investigated cytokines from intracellular content, direct cellular secretions, and extracellular vesicles from adult adipose-derived stem cells isolated from three distinct anatomical locations: abdomen, thigh, and chin. The cells were cultured investigated using live cell microscopy, cytokine assays, and bioinformatics analysis. The cytokines quantified and examined from each sample type showed a distinct difference between niche areas and sample types. The varying levels of TNF-alpha, IL-6 and IL-8 cytokines were shown to play a crucial role in signalling pathways such as MAPK, ERK1/2 and JAK-STAT in cells. On the other hand, the chemotactic cytokines IL-1rn, Eotaxin, IP-10 and MCP-1 showed the most prominent changes across extracellular vesicles with roles in noncanonical signalling. By examining the local and tangential roles of cytokines in stem cells, their roles in signalling and in regenerative mechanisms may be further understood.

Dense connectomic mapping of neuronal circuits is limited by the time and effort required to analyze 3D electron microscopy (EM) datasets. Algorithms designed to automate image segmentation suffer from substantial error rates and require significant manual error correction. Any improvement in segmentation error rates would therefore directly reduce the time required to analyze 3D EM data. We explored preserving extracellular space (ECS) during chemical tissue fixation to improve the ability to segment neurites and to identify synaptic contacts. ECS preserved tissue is easier to segment using machine learning algorithms, leading to significantly reduced error rates. In addition, we observed that electrical synapses are readily identified in ECS preserved tissue. Finally, we determined that antibodies penetrate deep into ECS preserved tissue with only minimal permeabilization, thereby enabling correlated light microscopy (LM) and EM studies. We conclude that preservation of ECS benefits multiple aspects of the connectomic analysis of neural circuits.

Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action1-3. ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol4-6 and thereby contribute to essential cellular processes, adaptive immunity and multidrug resistance7,8. Despite their importance, the coupling of nucleotide binding, hydrolysis and release to the conformational dynamics of these proteins remains poorly resolved, especially for heterodimeric and/or asymmetric ABC exporters that are abundant in humans. Here we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in a lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations-one of them with a bound peptide substrate-and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains and a closed extracellular gate. By decreasing the rate of ATP hydrolysis, we could capture an outward-facing (OF) open conformation-an otherwise transient state vulnerable to substrate re-entry. The ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent; both comprise co-existing OF conformations with open and closed extracellular gates. By contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ATP and ADP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental steps of the substrate translocation cycle of asymmetric ABC transporters.

Cytoplasmatic calcium concentrations are elevated two to three fold during cerebral ischemia. In order to determine the role of calcium-release from intracellular stores vs. calcium entry from the extracellular space, intracellular stores were depleted by the use of thapsigargin and calcium was removed from the incubation fluid prior to energy deprivation (ED). CA 1 pyramidal neurons in hippocampal rat slices were filled with a 1:2 mixture of Fluo-3 and Fura Red by intracellular injection. The neurons were visualized in a Confocal Laser Scanning Microscope (CLSM) and the fluorescence ratio from the probe mixture was used to quantify the calcium concentration. Intracellular calcium concentration was monitored before and during ED. The intracellular calcium concentration was 55 nM prior to ED and increased to 25 microM during ED. The resting levels were the same in the experimental groups, but the increase during ED was significantly lower in the intervention groups. The increase in the calcium free group was to 1 microM and in the thapsigargin group to 5 microM. In the last experimental group, thapsigargin treatment and removal of extracellular calcium, the intracellular calcium increased to 630 nM. These results demonstrate that the increased intracellular calcium seen during ED originates from several sources. Calcium-release from intracellular stores may be of major importance in calcium-related neuronal injury during cerebral ischemia.

Acyl-CoA binding protein (ACBP), also called diazepam-binding inhibitor (DBI), is a ubiquitous protein that can be secreted from cells by an unconventional pathway. Depending on its levels and on its subcellular localization, ACBP/DBI can regulate lipid metabolism. Several studies have shown that ACBP/DBI is secreted by an autophagy-dependent mechanism, positioning this catabolic pathway as the mechanism that controls lipid metabolism through the intracellular modulation of the levels of this protein. Autophagy is activated, among other stimuli, when cells have increased energy requirements; this causes a drop in the intracellular ACBP/DBI levels due to its release into the extracellular space and triggers an increase in the lipid catabolism. Conversely, when autophagy is inhibited, during pathological (obesity) or physiological (after-meal) situations, the intracellular levels of ACBP/DBI increase resulting in the activation of lipid anabolism, this effect has been demonstrated to be the link between obesity and autophagy inhibition. Here, we detail three different protocols for the detection of the ACBP/DBI levels by immunofluorescence, image flow cytometry or immunoblot techniques, which allow the quantification of ACBP/DBI levels and, indirectly, its autophagy-dependent release.

Glutathione is present in millimolar concentrations in the cell, but its relative distribution among cellular compartments remains elusive. We have chosen the endoplasmic reticulum (ER) as an example organelle to study compartment-specific glutathione levels. Using a glutaredoxin sensor (sCGrx1pER), which rapidly and specifically equilibrates with the reduced glutathione (GSH)-glutathione disulfide (GSSG) redox couple with known equilibrium constant, we showed that the [GSH]:[GSSG] ratio in the ER of intact HeLa cells is less than 7:1. Taking into consideration the previously determined value for [GSH](2):[GSSG] in the ER of 83 mM, this translates into a total glutathione concentration in the ER ([GStot]=[GSH]+2[GSSG]) of greater than 15 mM. Since the integrated, intracellular [GStot] was measured as ~7 mM, we conclude the existence of a [GStot] gradient across the ER membrane. A possible homeostatic mechanism by which cytosol-derived glutathione is trapped in the ER is discussed. We propose a high [GStot] as a distinguishing feature of the ER environment compared to the extracellular space.

Gravity-sensitive cellular responses are regularly observed in both specialized and nonspecialized cells. One potential mechanism for this sensitivity is a changing viscosity of the intracellular organelles. Here, we report a novel, to our knowledge, viscosity-sensitive molecular rotor based on mesosubstituted boron-dipyrrin used to investigate the response of viscosity of cellular membranes to hypergravity conditions created at the large diameter centrifuge at the European Space Agency Technology Centre. Mouse osteoblastic (MC3T3-E1) and endothelial (human umbilical vein endothelial cell) cell lines were tested, and an increase in viscosity was found with increasing hypergravity loading. This response is thought to be primarily biologically driven, with the potential for a small, instantaneous physical mechanism also contributing to the observed effect. This work provides the first, to our knowledge, quantitative data for cellular viscosity changes under hypergravity, up to 15 × g.

Ascorbate is an important element of a variety of cellular processes including the control of reactive oxygen species levels. Since reactive oxygen species are implicated as a key factor in tumorigenesis and antitumor therapy, the injection of a large amount of ascorbate is considered beneficial in cancer therapy. Recent studies have shown that ascorbate can cross the plasma membrane through passive diffusion. In contrast to absorption by active transport, which is facilitated by transport proteins (SVCT1 and SVCT2). The passive diffusion of a weak acid across membranes depends on the electrostatic potential and the pH gradients. This has been used to construct a new theoretical model capable of providing steady-state ascorbate concentration in the intracellular space and evaluating the time needed to reach it. The main conclusion of the analysis is that the steady-state intracellular ascorbate concentration weakly depends on its serum concentration but requires days of exposure to saturate. Based on these findings, it can be hypothesized that extended oral ascorbate delivery is possibly more effective than a short intravenous infusion of high ascorbate quantities.

Although protein-protein interactions (PPIs) have emerged as the basis of potential new therapeutic approaches, targeting intracellular PPIs with small molecule inhibitors is conventionally considered highly challenging. Driven by increasing research efforts, success rates have increased significantly in recent years. In this study, we analyze the physicochemical properties of 9351 non-redundant inhibitors present in the iPPI-DB and TIMBAL databases to define a computational model for active compounds acting against PPI targets. Principle component analysis (PCA) and k-means clustering were used to identify plausible PPI targets in regions of interest in the active group in the chemical space between active and inactive iPPI compounds. Notably, the uniquely defined active group exhibited distinct differences in activity compared with other active compounds. These results demonstrate that active compounds with regions of interest in the chemical space may be expected to provide insights into potential PPI inhibitors for particular protein targets.

Recent studies have strongly indicated that the classic circulation model of cerebrospinal fluid (CSF) is no longer valid. The production of CSF is not only dependent on the choroid plexus but also on water flux in the peri-capillary (Virchow Robin) space. Historically, CSF flow through the Virchow Robin space is known as interstitial flow, the physiological significance of which is now fully understood. This article briefly reviews the modern concept of CSF physiology and the Virchow-Robin space, in particular its functionalities critical for central nervous system neural activities. Water influx into the Virchow Robin space and, hence, interstitial flow is regulated by aquaporin-4 (AQP-4) localized in the endfeet of astrocytes, connecting the intracellular cytosolic fluid space of astrocytes and the Virchow Robin space. Interstitial flow has a functionality equivalent to systemic lymphatics, on which clearance of β-amyloid is strongly dependent. Autoregulation of brain blood flow serves to maintain a constant inner capillary fluid pressure, allowing fluid pressure of the Virchow Robin space to regulate regional cerebral blood flow (rCBF) based on AQP-4 gating. Excess heat produced by neural activities is effectively removed from the area of activation by increased rCBF by closing AQP-4 channels. This neural flow coupling (NFC) is likely mediated by heat generated proton channels.

Increasingly, renewable energy comprises a larger share of global energy production. Across the western United States, public lands are being developed to support renewable energy production. Where there are conflicts with threatened or endangered species, translocation can be used in an attempt to mitigate negative effects. For the threatened Mojave desert tortoise (Gopherus agassizii), we sought to compare habitat- and space-use patterns between short-distance translocated, resident, and control groups. We tested for differences in home range size based on utilization distributions and used linear mixed-effects models to compare space-use intensity, while controlling for demographic and environmental variables. In addition, we examined mean movement distances as well as home range overlap between years and for male and female tortoises in each study group. During the first active season post-translocation, home range size was greater and space-use intensity was lower for translocated tortoises than resident and control groups. These patterns were not present in the second season. In both years, there was no difference in home range size or space-use intensity between control and resident groups. Translocation typically resulted in one active season of questing followed by a second active season characterized by space-use patterns that were indistinguishable from control tortoises. Across both years, the number of times a tortoise was found in a burrow was positively related to greater space-use intensity. Minimizing the time required for translocated tortoises to exhibit patterns similar to non-translocated individuals may have strong implications for conservation by reducing exposure to adverse environmental conditions and predation. With ongoing development, our results can be used to guide future efforts aimed at understanding how translocation strategies influence patterns of animal space use.

Intracellular transport is predominantly heterogeneous in both time and space, exhibiting varying non-Brownian behavior. Characterization of this movement through averaging methods over an ensemble of trajectories or over the course of a single trajectory often fails to capture this heterogeneity. Here, we developed a deep learning feedforward neural network trained on fractional Brownian motion, providing a novel, accurate and efficient method for resolving heterogeneous behavior of intracellular transport in space and time. The neural network requires significantly fewer data points compared to established methods. This enables robust estimation of Hurst exponents for very short time series data, making possible direct, dynamic segmentation and analysis of experimental tracks of rapidly moving cellular structures such as endosomes and lysosomes. By using this analysis, fractional Brownian motion with a stochastic Hurst exponent was used to interpret, for the first time, anomalous intracellular dynamics, revealing unexpected differences in behavior between closely related endocytic organelles.

The importance of innate immunity in cancer is increasingly being recognized with recent reports suggesting tumor cell-intrinsic intracellular functions for innate immunity proteins. However, such functions are often poorly understood, and it is unclear whether these are affected by patient-specific mutations. Here, we show that C4b-binding protein alpha chain (C4BPA), typically thought to reside in the extracellular space, is expressed intracellularly in cancer cells, where it interacts with the NF-κB family member RelA and regulates apoptosis. Interestingly, intracellular C4BPA expression is regulated in a stress- and mutation-dependent manner and C4BPA mutations are associated with improved cancer survival outcome. Using cell lines harboring patient-specific C4BPA mutations, we show that increasing intracellular C4BPA levels correlate with sensitivity to oxaliplatin-induced apoptosis in vitro and in vivo. Mechanistically, sensitive C4BPA mutants display increased IκBα expression and increased inhibitory IκBα-RelA complex stability. These data suggest a non-canonical intracellular role for C4BPA in regulating NF-κB-dependent apoptosis.

Galectin-3 is a carbohydrate binding protein which has important roles in cancer and immunity. Potent galectin-3 inhibitors have been synthesized, for experimental purposes and potential clinical use. As galectin-3 is implicated in both intra- and extracellular activities, permeability of galectin-3 inhibitors is an important parameter determining biological effects. We compared the cellular uptake of galectin-3 inhibitors and their potency in the intracellular or extracellular space. The inhibitors differed in their polar surface area (PSA), but had similar affinities for galectin-3. Using a well-established permeability assay, we confirmed that the uptake was significantly higher for the inhibitor with the lowest PSA, as expected. To analyze intracellular activity of the inhibitors, we developed a novel assay based on galectin-3 accumulation around damaged intracellular vesicles. The results show striking differences between the inhibitors intracellular potency, correlating with their PSAs. To test extracellular activity of the inhibitors, we analyzed their potency to block binding of galectin-3 to cell surfaces. All inhibitors were equally able to block galectin-3 binding to cells and this was proportional to their affinity for galectin-3. These inhibitors may serve as useful tools in exploring biological roles of galectin-3 and may further our understanding of intracellular versus extracellular roles of galectin-3.

Liver cells possess store-operated Ca2+ channels (SOCs) with a high selectivity for Ca2+ compared with Na+, and several types of intracellular messenger-activated non-selective cation channels with a lower selectivity for Ca2+ (NSCCs). The main role of SOCs is thought to be in refilling depleted endoplasmic reticulum Ca2+ stores [Cell Calcium 7 (1986) 1]. NSCCs may be involved in refilling intracellular stores but are also thought to have other roles in regulating the cytoplasmic-free Ca2+ and Na+ concentrations. The ability of SOCs to refill the endoplasmic reticulum Ca2+ stores in hepatocytes has not previously been compared with that of NSCCs. The aim of the present studies was to compare the ability of SOCs and maitotoxin-activated NSCCs to refill the endoplasmic reticulum in rat hepatocytes. The experiments were performed using fura-2FF and fura-2 to monitor the free Ca2+ concentrations in the endoplasmic reticulum and cytoplasmic space, respectively, a Ca2+ add-back protocol, and 2-aminoethyl diphenylborate (2-APB) to inhibit Ca2+ inflow through SOCs. In cells treated with 2,5-di-t-butylhydroquinone (DBHQ) or vasopressin to deplete the endoplasmic reticulum Ca2+ stores, then washed to remove DBHQ or vasopressin, the addition of Ca2+ caused a substantial increase in the concentration of Ca2+ in the endoplasmic reticulum and cytoplasmic space due to the activation of SOCs. These increases were inhibited 80% by 2-APB, indicating that Ca2+ inflow is predominantly through SOCs. In the presence of 2-APB (to block SOCs), maitotoxin induced a substantial increase in [Ca2+](cyt), but only a modest and slower increase in [Ca2+](er). Under these conditions, Ca2+ inflow is predominantly through maitotoxin-activated NSCCs. It is concluded that SOCs are more effective than maitotoxin-activated NSCCs in refilling the endoplasmic reticulum Ca2+ stores. The previously developed concept of a specific role for SOCs in refilling the endoplasmic reticulum is consistent with the results reported here.