Cytoplasmic Diffusion Research Articles

Single-cell O2 exchange imaging shows that cytoplasmic diffusion is a dominant barrier to efficient gas transport in red blood cells

Disorders of oxygen transport are commonly attributed to inadequate carrying capacity (anemia) but may also relate to inefficient gas exchange by red blood cells (RBCs), a process that is poorly characterized yet assumed to be rapid. Without direct measurements of gas exchange at the single-cell level, the barriers to O2 transport and their relationship with hematological disorders remain ill defined. We developed a method to track the flow of O2 in individual RBCs by combining ultrarapid solution switching (to manipulate gas tension) with single-cell O2 saturation fluorescence microscopy. O2 unloading from RBCs was considerably slower than previously estimated in acellular hemoglobin solutions, indicating the presence of diffusional barriers in intact cells. Rate-limiting diffusion across cytoplasm was demonstrated by osmotically induced changes to hemoglobin concentration (i.e., diffusive tortuosity) and cell size (i.e., diffusion pathlength) and by comparing wild-type cells with hemoglobin H (HbH) thalassemia (shorter pathlength and reduced tortuosity) and hereditary spherocytosis (HS; expanded pathlength). Analysis of the distribution of O2 unloading rates in HS RBCs identified a subpopulation of spherocytes with greatly impaired gas exchange. Tortuosity imposed by hemoglobin was verified by demonstrating restricted diffusivity of CO2, an acidic gas, from the dissipative spread of photolytically uncaged H+ ions across cytoplasm. Our findings indicate that cytoplasmic diffusion, determined by pathlength and tortuosity, is a major barrier to efficient gas handling by RBCs. Consequently, changes in RBC shape and hemoglobin concentration, which are common manifestations of hematological disorders, can have hitherto unrecognized and clinically significant implications on gas exchange.

Accelerating itaconic acid production by increasing membrane permeability of whole-cell biocatalyst based on a psychrophilic bacterium Shewanella livingstonensis Ac10

Whole-cell biocatalysts have numerous advantages including ease of preparation and coenzyme recovery over purified industrially used enzymes. However, the cell membrane can occasionally hinder cytoplasmic diffusion of the substrate, resulting in reduced biotransformation efficiency. Psychrophiles can grow and reproduce at low temperatures; their cell membranes are highly flexible, and their permeability can be improved via heat treatment at a moderate temperature. The aim of this study was to generate a psychrophile-based simple biocatalyst (PSCats) using Shewanella livingstonensis Ac10. This biocatalyst contained two enzymes that were heterologously expressed and converted citric acid to itaconic acid, thereby serving as a potential platform replacing the petroleum-based counterparts. The efficiency of the biocatalyst was increased via heat treatment at 45 °C for 15 min, and itaconic acid productivity of the cells after heat treatment (1.41 g/L/h) was increased around 6-fold in comparison with those without heat treatment (0.22 g/L/h). A large part of the productivity remained (67.3 %) when the cells were reused for 5 times (10 h for each reaction). Therefore, the potential of this heat-permeabilized psychrophile host to increase the productivity of whole-cell biocatalyst was proved; however, further research is necessary to understand the underlying mechanism.

Distinct Kinetics in Electrophoretic Extraction of Cytoplasmic RNA from Single Cells.

The physical fractionation of cytoplasmic versus nuclear components of cells is a key step for studying the subcellular localization of molecules. The application of an electric field is an emerging method for subcellular fractionation of proteins and nucleic acids from single cells. However, the multibiophysical process that involves electrical lysis of cytoplasmic membranes, electrophoresis, and diffusion of charged molecules remains unclear. Here we study RNA dynamics in single cells during the electrophoretic extraction via a microfluidic system that enables stringent fractionation of the subcellular components leveraging a focused electric field. We identified two distinct kinetics in the extraction of RNA molecules, which were respectively associated with soluble RNA and mitochondrial RNA. We show that the extraction kinetics of soluble RNA is dominated by electrophoresis over diffusion and has a time constant of 0.15 s. Interestingly, the extraction of mitochondrial RNA showed unexpected heterogeneity in the extraction with slower kinetics (3.8 s), whilereproducibly resulting in the extraction of 98.9% ± 2% after 40 s. Together, we uncover that the microfluidic system uniquely offers length bias-free fractionation of RNA molecules for quantitative analysis of correlations among subcellular compartments by exploiting the homogeneous electrophoretic properties of RNA.

The Role of Cell Membrane Information Reception, Processing, and Communication in the Structure and Function of Multicellular Tissue.

Investigations of information dynamics in eukaryotic cells focus almost exclusively on heritable information in the genome. Gene networks are modeled as “central processors” that receive, analyze, and respond to intracellular and extracellular signals with the nucleus described as a cell’s control center. Here, we present a model in which cellular information is a distributed system that includes non-genomic information processing in the cell membrane that may quantitatively exceed that of the genome. Within this model, the nucleus largely acts a source of macromolecules and processes information needed to synchronize their production with temporal variations in demand. However, the nucleus cannot produce microsecond responses to acute, life-threatening perturbations and cannot spatially resolve incoming signals or direct macromolecules to the cellular regions where they are needed. In contrast, the cell membrane, as the interface with its environment, can rapidly detect, process, and respond to external threats and opportunities through the large amounts of potential information encoded within the transmembrane ion gradient. Our model proposes environmental information is detected by specialized protein gates within ion-specific transmembrane channels. When the gate receives a specific environmental signal, the ion channel opens and the received information is communicated into the cell via flow of a specific ion species (i.e., K+, Na+, Cl−, Ca2+, Mg2+) along electrochemical gradients. The fluctuation of an ion concentration within the cytoplasm adjacent to the membrane channel can elicit an immediate, local response by altering the location and function of peripheral membrane proteins. Signals that affect a larger surface area of the cell membrane and/or persist over a prolonged time period will produce similarly cytoplasmic changes on larger spatial and time scales. We propose that as the amplitude, spatial extent, and duration of changes in cytoplasmic ion concentrations increase, the information can be communicated to the nucleus and other intracellular structure through ion flows along elements of the cytoskeleton to the centrosome (via microtubules) or proteins in the nuclear membrane (via microfilaments). These dynamics add spatial and temporal context to the more well-recognized information communication from the cell membrane to the nucleus following ligand binding to membrane receptors. Here, the signal is transmitted and amplified through transduction by the canonical molecular (e.g., Mitogen Activated Protein Kinases (MAPK) pathways. Cytoplasmic diffusion allows this information to be broadly distributed to intracellular organelles but at the cost of loss of spatial and temporal information also contained in ligand binding.

Assessment of DNA Topoisomerase I Unwinding Activity, Radical Scavenging Capacity, and Inhibition of Breast Cancer Cell Viability of N-alkyl-acridones and N,N'-dialkyl-9,9'-biacridylidenes.

The anticancer activity of acridone derivatives has attracted increasing interest, therefore, a variety of substituted analogs belonging to this family have been developed and evaluated for their anti-cancer properties. A series of N-alkyl-acridones 1–6 and N,N′-dialkyl-9,9′-biacridylidenes 7–12 with variable alkyl chains were examined for their topoisomerase I activity at neutral and acidic conditions as well as for their binding capacity to calf thymus and possible radical trapping antioxidant activity. It was found that at a neutral pH, topoisomerase I activity of both classes of compounds was similar, while under acidic conditions, enhanced intercalation was observed. N-alkyl-acridone derivatives 1–6 exhibited stronger, dose-dependent, cytotoxic activity against MCF-7 human breast epithelial cancer cells than N,N′-dialkyl-9,9′-biacridylidenes 7–12, revealing that conjugation of the heteroaromatic system plays a significant role on the effective distribution of the compound in the intracellular environment. Cellular investigation of long alkyl derivatives against cell migration exhibited 40–50% wound healing effects and cytoplasm diffusion, while compounds with shorter alkyl chains were accumulated both in the nucleus and cytoplasm. All N,N′-dialkyl-9,9′-biacridylidenes showed unexpected high scavenging activity towards DPPH or ABTS radicals which may be explained by higher stabilization of radical cations by the extended conjugation of heteroaromatic ring system.

All-in-One Synchronized DNA Nanodevices Facilitating Multiplexed Cell Imaging.

Multifunctional DNA nanodevices perform ever more tasks with applications ranging from in vitro biomarker detection to in situ cell imaging. However, most developed ones consist of a series of split building blocks, which suffer from asynchronous behaviors in complicated cellular microenvironments (endocytosis pathway, diffusion-limited cytoplasm, etc.), causing the loss of stoichiometric information and additional postassembly processes. Herein, we constructed all-in-one DNA nanodevices to achieve synchronous multiplexed imaging. All DNA components, including two sets of probe modules (each containing target-specific walkers, i.e., hairpin tracks with chemically damaged bases), are modified on individual gold nanoparticles. This design not only enables their integrated internalization into cells, circumventing inhomogeneous distribution of different building blocks and increasing the local concentrations of the interacting modules, but also avoids the impact of stochastic diffusion in viscous cytoplasm. A couple of intracellular enzymes in situ actuate the synchronized motion of the modules, all on-particle, after specific recognition of intracellular targets (such as RNAs and proteins), thus facilitating synchronized, multiplexed cell imaging. Finally, the proposed all-in-one nanodevices were successfully applied to monitor intracellular microRNA-21 and telomerase expression levels. The flexible design can be extended to detect other cytoplasmic molecules and monitor related pathways by simply changing the sequences.

NIR-Light-Triggered Anticancer Strategy for Dual-Modality Imaging-Guided Combination Therapy via a Bioinspired Hybrid PLGA Nanoplatform.

A promising approach toward cancer therapy is expected to integrate imaging and therapeutic agents into a versatile nanocarrier for achieving improved antitumor efficacy and reducing the side effects of conventional chemotherapy. Herein, we designed a poly(d,l-lactic- co-glycolic acid) (PLGA)-based theranostic nanoplatform using the double emulsion solvent evaporation method (W/O/W), which is associated with bovine serum albumin (BSA) modifications, to codeliver indocyanine green (ICG), a widely used near-infrared (NIR) dye, and doxorubicin (Dox), a chemotherapeutic drug, for dual-modality imaging-guided chemo-photothermal combination cancer therapy. The resultant ICG/Dox co-loaded hybrid PLGA nanoparticles (denoted as IDPNs) had a diameter of around 200 nm and exhibited excellent monodispersity, fluorescence/size stability, and biocompatibility. It was confirmed that IDPNs displayed a photothermal effect and that the heat induced faster release of Dox, which led to enhanced drug accumulation in cells and was followed by their efficient escape from the lysosomes into the cytoplasm and drug diffusion into the nucleus, resulting in a chemo-photothermal combinatorial therapeutic effect in vitro. Moreover, the IDPNs exhibited a high ability to accumulate in tumor tissue, owing to the enhanced permeability and retention (EPR) effect, and could realize real-time fluorescence/photoacoustic imaging of solid tumors with a high spatial resolution. In addition, the exposure of tumor regions to NIR irradiation could enhance the tumor penetration ability of IDPNs, almost eradicating subcutaneous tumors. In addition, the inhibition rate of IDPNs used in combination with laser irradiation against EMT-6 tumors in tumor-bearing nude mice (chemo-photothermal therapy) was approximately 95.6%, which was much higher than that for chemo- or photothermal treatment alone. Our study validated the fact that the use of well-defined IDPNs with NIR laser treatment could be a promising strategy for the early diagnosis and passive tumor-targeted chemo-photothermal therapy for cancer.

Subdiffusion of loci and cytoplasmic particles are different in compressed Escherichia coli cells

The complex physical nature of the bacterial intracellular environment remains largely unknown, and has relevance for key biochemical and biological processes of the cell. Although recent work has addressed the role of non-equilibrium sources of activity and crowding, the consequences of mechanical perturbations are relatively less explored. Here we use a microfabricated valve system to track both fluorescently labeled chromosomal loci and cytoplasmic particles in Escherichia coli cells shortly after applying a compressive force, observing the response on time scales that are too sudden to allow for biochemical response from the cell. Cytoplasmic diffusion slows markedly on compression but the exponent governing the growth of the ensemble-averaged mean-squared displacement of cytoplasmic particles is unaffected. In contrast, the corresponding exponent for DNA loci changes significantly. These results suggest that DNA elasticity and nucleoid organization play a more important role in loci subdiffusion than cytoplasmic viscoelasticity under such short time scales.

Non-specific interactions govern cytosolic diffusion of nanosized objects in mammalian cells.

The diffusivity of macromolecules in the cytoplasm of eukaryotic cells varies over orders of magnitude and dictates the kinetics of cellular processes. However, a general description that associates the Brownian or anomalous nature of intracellular diffusion to the architectural and biochemical properties of the cytoplasm has not been achieved. Here we measure the mobility of individual fluorescent nanoparticles in living mammalian cells to obtain a comprehensive analysis of cytoplasmic diffusion. We identify a correlation between tracer size, its biochemical nature and its mobility. Inert particles with size equal or below 50 nm behave as Brownian particles diffusing in a medium of low viscosity with negligible effects of molecular crowding. Increasing the strength of non-specific interactions of the nanoparticles within the cytoplasm gradually reduces their mobility and leads to subdiffusive behaviour. These experimental observations and the transition from Brownian to subdiffusive motion can be captured in a minimal phenomenological model.

Characterization of Cytoskeletal Pore Size Using Quantum Dots

Cytoskeletal pore size is a key factor in determining the intracellular molecular transportation, yet it is technically challenging to investigate correlations between the cytoskeletal pore size and cytoplasmic diffusion. Here, we report the application of quantum dots (QD) to measure the cytoskeletal pore size. QDs are first transfected into a cell, leading to different sizes of QD-encapsulating vesicles. The distribution of QD vesicles in cells reflects the cytoskeletal pore size. Further, we can predict the cytosolic viscosity from the measured pore size using the poroelasticity scaling theory. This predicted cytosolic viscosity is verified by measuring Brownian motions of the QDs in cells and transforming the motion characteristics into cytosolic viscosity via the Stocks–Einstein relation. This strategy also provides a practical method for quantifying the cytoskeletal pore sizes. Together, this research demonstrates the efficacy of applying QDs in further studies of intracellular physical properties that play key roles in intracellular molecular transports.

Design, characterization and in vivo functioning of a light-dependent histidine protein kinase in the yeast Saccharomyces cerevisiae

Helical alignment of the α-helical linker of the LOV (light-oxygen-voltage) domain of YtvA from Bacillus subtilis with the α-helical linker of the histidine-protein kinase domain of the Sln1 kinase of the phospho-relay system for osmoregulation of Saccharomyces cerevisiae has been used to construct a light-modulatable histidine protein kinase. In vitro, illumination with blue light inhibits both the ATP-dependent phosphorylation of this hybrid kinase, as well as the phosphoryl transfer to Ypd1, the phosphoryl transfer domain of the Sln1 system. The helical alignment was carried out with conservation of the complete Jα helix of YtvA, as well as of the phosphorylatable histidine residue of the Sln1 kinase, with conservation of the hepta-helical motive of coiled-coil structures, recognizable in the helices of the two separate, constituent, proteins. Introduction of the gene encoding this hybrid histidine protein kinase into cells of S. cerevisiae in which the endogenous Sln1 kinase had been deleted, allowed us to modulate gene expression in the yeast cells with (blue) light. This was first demonstrated via the light-induced alteration of the expression level of the mannosyl-transferase OCH1, via a translational-fusion approach. As expected, illumination decreased the expression level of OCH1; the steady state decrease in saturating levels of blue light was about 40%. To visualize the in vivo functionality of this light-dependent regulation system, we fused the green fluorescent protein (GFP) to another regulatory protein, HOG1, which is also responsive to the Sln1 kinase. HOG1 is phosphorylated by the MAP-kinase-kinase Pbs2, which in turn is under control of the Sln1 kinase, via the phosphoryl transfer domain Ypd1. Fluorescence microscopy was used to show that illumination of cells that contained the combination of the hybrid kinase and the HOG1::GFP fusion protein, led to a persistent increase in the level of nuclear accumulation of HOG1, in contrast to salt stress, which—as expected—showed the well-characterized transient response. The system described in this study will be valuable in future studies on the role of cytoplasmic diffusion in signal transduction in eukaryotic cells.

Compressive Stress Stalls Growth and Decrease Cytoplasmic Diffusion

Compressive Stress Stalls Growth and Decrease Cytoplasmic Diffusion

Recruitment of UvrBC complexes to UV-induced damage in the absence of UvrA increases cell survival.

Nucleotide excision repair (NER) is the primary mechanism for removal of ultraviolet light (UV)-induced DNA photoproducts and is mechanistically conserved across all kingdoms of life. Bacterial NER involves damage recognition by UvrA2 and UvrB, followed by UvrC-mediated incision either side of the lesion. Here, using a combination of in vitro and in vivo single-molecule studies we show that a UvrBC complex is capable of lesion identification in the absence of UvrA. Single-molecule analysis of eGFP-labelled UvrB and UvrC in living cells showed that UV damage caused these proteins to switch from cytoplasmic diffusion to stable complexes on DNA. Surprisingly, ectopic expression of UvrC in a uvrA deleted strain increased UV survival. These data provide evidence for a previously unrealized mechanism of survival that can occur through direct lesion recognition by a UvrBC complex.

CBP-mediated SMN acetylation modulates Cajal body biogenesis and the cytoplasmic targeting of SMN.

The survival of motor neuron (SMN) protein plays an essential role in the biogenesis of spliceosomal snRNPs and the molecular assembly of Cajal bodies (CBs). Deletion of or mutations in the SMN1 gene cause spinal muscular atrophy (SMA) with degeneration and loss of motor neurons. Reduced SMN levels in SMA lead to deficient snRNP biogenesis with consequent splicing pathology. Here, we demonstrate that SMN is a novel and specific target of the acetyltransferase CBP (CREB-binding protein). Furthermore, we identify lysine (K) 119 as the main acetylation site in SMN. Importantly, SMN acetylation enhances its cytoplasmic localization, causes depletion of CBs, and reduces the accumulation of snRNPs in nuclear speckles. In contrast, the acetylation-deficient SMNK119R mutant promotes formation of CBs and a novel category of promyelocytic leukemia (PML) bodies enriched in this protein. Acetylation increases the half-life of SMN protein, reduces its cytoplasmic diffusion rate and modifies its interactome. Hence, SMN acetylation leads to its dysfunction, which explains the ineffectiveness of HDAC (histone deacetylases) inhibitors in SMA therapy despite their potential to increase SMN levels.

Diffusive Behavior of Mismatch Repair Protein MSH2 in Cells at Different Stages of Cancer

Cellular interiors present media that are very complex as they encompass a mixture of proteins, organelles, viscoelastic filaments, and chromatin structures in the nuclear interior. The behavior of proteins in such environments reflects the cellular complexity. Here we present the results of studies on the mobility, or diffusion, of free eGFP proteins, and of the important nuclear mismatch repair protein, MSH2. MSH2 was labeled with eGFP and the diffusion of both the free eGFP and MSH2-eGFP was measured using Fluorescence Recovery After Photobleaching (FRAP) on four distinct cell types that act as a model for the neoplastic cancer transformation of human mammary epithelial cells from non-cancerous to metastatic. We obtained several interesting results, such as a significant change in behavior for the first stage of cancer (immortal) compared to other stages. We also observed a significantly lower value for MSH2 diffusion (in both cytoplasm and nucleus) than what would be expected by scaling from the eGFP values (using appropriate size ratios of the two proteins). Diffusion coefficients ranged from 14 to 24 μm2/s for EGFP and from 3 to 7 μm2/s for EGFP-MSH2. This material is based upon work supported by the National Science Foundation under Grant Number 1106105 (KB, KS, and MG). We also thank Glen Marrs, director of the WFU Microscopic Imaging Core Facility, and Anita McCauley, for their help with the confocal microscopy setup.

Protein Charge and Ion Concentration Affect Protein Diffusion in the Prokaryotic Cytoplasm

Contrary to eukaryotic cells, both bacteria and archaea (mostly) lack active cytoplasmic transport. The encounter rate between proteins in the cytoplasm thus depends (almost) exclusively on lateral diffusion. Previously, it was found that the cytoplasmic protein diffusion coefficient drops with an increase of (1) its molecular weight and (2) cytoplasmic excluded volume. Here we show the dependence of the protein diffusion coefficient on the strength of binding interactions. We have done FRAP measurements to obtain diffusion coefficients of surface-modified GFPs in Escherichia coli, Lactococcus lactis and Haloferax volcanii. The tested variants of GFP vary in net charge from −30 to +25. The examined organisms differ in their cytoplasmic ion concentration. The GFP diffusion coefficient drops with increasing positive charge, up to two orders of magnitude. This is caused by binding of the GFPs to ribosomes. The drop in the diffusion coefficient decreases with increased cytoplasmic ion concentration. Our findings suggest a limit to what the surface charge of a protein can be in the cytoplasm of a given cell.

Hindered cytoplasmic diffusion of inositol trisphosphate restricts its cellular range of action.

The range of action of intracellular messengers is determined by their rates of diffusion and degradation. Previous measurements in oocyte cytoplasmic extracts indicated that the Ca2+-liberating second messenger inositol trisphosphate (IP3) diffuses with a coefficient (~280 μm2 s-1) similar to that in water, corresponding to a range of action of ~25 μm. Consequently, IP3 is generally considered a "global" cellular messenger. We reexamined this issue by measuring local IP3-evoked Ca2+ puffs to monitor IP3 diffusing from spot photorelease in neuroblastoma cells. Fitting these data by numerical simulations yielded a diffusion coefficient (≤10 μm2 s-1) about 30-fold slower than that previously reported. We propose that diffusion of IP3 in mammalian cells is hindered by binding to immobile, functionally inactive receptors that were diluted in oocyte extracts. The predicted range of action of IP3 (<5 μm) is thus smaller than the size of typical mammalian cells, indicating that IP3 should better be considered as a local rather than a global cellular messenger.

Red blood cell thickness is evolutionarily constrained by slow, hemoglobin-restricted diffusion in cytoplasm.

During capillary transit, red blood cells (RBCs) must exchange large quantities of CO2 and O2 in typically less than one second, but the degree to which this is rate-limited by diffusion through cytoplasm is not known. Gas diffusivity is intuitively assumed to be fast and this would imply that the intracellular path-length, defined by RBC shape, is not a factor that could meaningfully compromise physiology. Here, we evaluated CO2 diffusivity (DCO2) in RBCs and related our results to cell shape. DCO2 inside RBCs was determined by fluorescence imaging of [H+] dynamics in cells under superfusion. This method is based on the principle that H+ diffusion is facilitated by CO2/HCO3− buffer and thus provides a read-out of DCO2. By imaging the spread of H+ ions from a photochemically-activated source (6-nitroveratraldehyde), DCO2 in human RBCs was calculated to be only 5% of the rate in water. Measurements on RBCs containing different hemoglobin concentrations demonstrated a halving of DCO2 with every 75 g/L increase in mean corpuscular hemoglobin concentration (MCHC). Thus, to compensate for highly-restricted cytoplasmic diffusion, RBC thickness must be reduced as appropriate for its MCHC. This can explain the inverse relationship between MCHC and RBC thickness determined from >250 animal species.

The residue at position 5 of the N-terminal region of Src and Fyn modulates their myristoylation, palmitoylation, and membrane interactions.

The interactions of Src family kinases (SFKs) with the plasma membrane are crucial for their activity. They depend on their fatty-acylated N-termini, containing N-myristate and either a polybasic cluster (in Src) or palmitoylation sites (e.g., Fyn). To investigate the roles of these moieties in SFK membrane association, we used fluorescence recovery after photobleaching beam-size analysis to study the membrane interactions of c-Src-GFP (green fluorescent protein) or Fyn-GFP fatty-acylation mutants. Our studies showed for the first time that the membrane association of Fyn is more stable than that of Src, an effect lost in a Fyn mutant lacking the palmitoylation sites. Unexpectedly, Src-S3C/S6C (containing cysteines at positions 3/6, which are palmitoylated in Fyn) exhibited fast cytoplasmic diffusion insensitive to palmitoylation inhibitors, suggesting defective fatty acylation. Further replacement of the charged Lys-5 by neutral Gln to resemble Fyn (Src-S3C/S6C/K5Q) restored Fyn-like membrane interactions, indicating that Lys-5 in the context of Src-S3C/S6C interferes with its myristoylation/palmitoylation. This was validated by direct myristoylation and palmitoylation studies, which indicated that the residue at position 5 regulates the membrane interactions of Src versus Fyn. Moreover, the palmitoylation levels correlated with targeting to detergent-resistant membranes (rafts) and to caveolin-1. Palmitoylation-dependent preferential containment of Fyn in rafts may contribute to its lower transformation potential.

Linear Channel Modeling and Error Analysis for Intra/Inter-Cellular Ca2+ Molecular Communication.

The use of intra/inter-cellular calcium ion (Ca2+) signaling for molecular communication (MC) is investigated in this paper. In particular, the elevation of the intracellular Ca2+ concentration upon the external excitation, i.e., Ca2+ wave generation, and the intercellular propagation of Ca2+ wave over consecutive cells are studied for information transmission. The main objective of this paper is to develop a linear channel model for intra/inter-cellular Ca2+ MC. In this context, the end-to-end Ca2+ MC system is studied under three blocks: the wave generation, the gap junctional (intercellular) propagation, and the intracellular propagation. The wave generation block captures the intracellular Ca2+ signaling pathway including the release of Ca2+ from the organelles and the buffers inside a cell, and the intake from the extracellular space. The gap junctional (intercellular) propagation block captures the Ca2+ transition through the gap junctions between the touching cells. The intracellular propagation block defines the effect of the cytoplasmic diffusion. Using the developed blocks for the different biophysical phenomena, the end-to-end channel gain and delay formulas are derived. Furthermore, the bit error probability is studied to reveal the impact of the detection threshold. This work provides the basis for the modeling, analysis and the design of Ca2+ MC systems.