The Secret Ballet Inside Multivesicular Bodies.

Lipid bilayers possess the capacity for self-assembly due to the amphipathic nature of lipid molecules, which have both hydrophobic and hydrophilic regions. When confined, lipid bilayers exhibit astonishing versatility in their forms, adopting diverse shapes that are challenging to observe through experimental means. Exploiting this adaptability, lipid structures motivate the development of bio-inspired mechanomaterials and integrated nanobio-interfaces that could seamlessly merge with biological entities, ultimately bridging the gap between synthetic and biological systems. In this work, we demonstrate how, in numerical simulations of multivesicular bodies, a fascinating evolution unfolds from an initial semblance of order toward states of higher entropy over time. We observe dynamic rearrangements in confined vesicles that reveal unexpected limit shapes of distinct geometric patterns. We identify five structures as the basic building blocks that systematically repeat under various conditions of size and composition. Moreover, we observe more complex and less frequent shapes that emerge in confined spaces. Our results provide insights into the dynamics of multivesicular systems, offering a richer understanding of how confined lipid bodies spontaneously self-organize.

Membrane Binding Strength vs Pore Formation Cost─What Drives the Membrane Permeation of Nanoparticles Coated with Cell-Penetrating Peptides?

Cell-penetrating peptides (CPPs) enable the transport of nanoparticles through cell membranes. Using molecular simulations, we conduct an in-depth investigation into the thermodynamic forces governing the passive translocation of CPP-coated nanoparticles across lipid bilayers, contrasting their behavior with that of bare particles to dissect the contribution of the peptides. Our analysis unveils a distinctive two-stage translocation mechanism, where the adsorption energy of the particles overcomes the cost of forming a hydrophilic transmembrane pore. Proper evaluation of the translocation mechanisms is only possible when using two reaction coordinates, in particular, one that explicitly includes the density of the lipids on the binding site of the particle. An analysis of adsorption and activation free energies in terms of a simple kinetic model provides a clearer understanding of the CPP effect. Experimental validation using nonendocytic cells confirms the superior membrane permeation of CPP-coated particles. Our findings have implications for the rational design of more efficient cell-permeating particles.

Kinetics of antigen cross-presentation assessed experimentally and by a model of the complete endomembrane system.

Dendritic cells (DCs) have a specialized endomembrane system capable of presenting exogenous antigens in the context of MHC class I (MHC-I) molecules. This process, named cross-presentation, is crucial to activate CD8+ T lymphocytes and initiate cytotoxic immune responses. In this report, we present an Agent-Based Model in combination with Ordinary Differential Equations with enough complexity to reproduce cross-presentation. The model embraces the secretory and endocytic pathways, in connection with the plasma membrane, the endoplasmic reticulum, and the cytosol. Key molecules required for cross-presentation were included as cargoes. In the simulations, the kinetics of MHC-I uptake and recycling, and cross-presentation accurately reproduced experimental values. The model proved to be a suitable tool to elaborate hypotheses and design experiments. In particular, the model predictions and the experimental results obtained indicate that the rate-limiting step in cross-presentation of soluble ovalbumin is MHC-I loading after proteasomal processing of the antigenic protein.

Enhanced Expansion and Reduced Kiss-and-Run Events in Fusion Pores Steered by Synaptotagmin-1 C2B Domains.

The fusion pore controls the release of exocytotic vesicle contents through a precise orchestration of lipids from the fusing membranes and proteins. There is a major lipid reorganization during the different stages in life of the fusion pore (membrane fusion, nucleation, and expansion) that can be scrutinized thermodynamically. In this work, using umbrella sampling simulations we describe the expansion of the fusion pore. We have calculated free energy profiles to drive a nascent, just nucleated, fusion pore to its expanded configuration. We have quantified the effects on the free energy of one and two Synaptotagmin-1 C2B domains in the cytosolic space. We show that C2B domains cumulatively reduce the cost for expansion, favoring the system to evolve toward full fusion. Finally, by conducting thousands of unbiased molecular dynamics simulations, we show that C2B domains significantly decrease the probability of kiss-and-run events.

Efficient Cholesterol Transport in Dendritic Cells Defines Optimal Exogenous Antigen Presentation and Toxoplasma gondii Proliferation.

Dendritic cells are the most powerful antigen-presenting cells of the immune system. They present exogenous antigens associated with Major Histocompatibility Complex (MHC) Class II molecules through the classical pathway to stimulate CD4+ T cells, or with MHC-I to activate CD8+ T lymphocytes through the cross-presentation pathway. DCs represent one of the main cellular targets during infection by Toxoplasma gondii. This intracellular parasite incorporates essential nutrients, such as cholesterol, to grow and proliferate inside a highly specialized organelle, the parasitophorous vacuole (PV). While doing so, T. gondii modulates the host immune response through multiple interactions with proteins and lipids. Cholesterol is an important cellular component that regulates cellular physiology at the structural and functional levels. Although different studies describe the relevance of cholesterol transport for exogenous antigen presentation, the molecular mechanism underlying this process is not defined. Here, we focus our study on the inhibitor U18666A, a drug widely used to arrest multivesicular bodies biogenesis that interrupts cholesterol trafficking and changes the lipid composition of intracellular membranes. Upon bone marrow-derived DC (BMDC) treatment with U18666A, we evidenced a drastic disruption in the ability to present exogenous soluble and particulate antigens to CD4+ and CD8+ T cells. Strikingly, the presentation of T. gondii-associated antigens and parasite proliferation were hampered in treated cells. However, neither antigen uptake nor BMDC viability was significantly affected by the U18666A treatment. By contrast, this drug altered the transport of MHC-I and MHC-II molecules to the plasma membrane. Since U18666A impairs the formation of MVBs, we analyzed in T. gondii infected BMDCs the ESCRT machinery responsible for the generation of intraluminal vesicles. We observed that different MVBs markers, including ESCRT proteins, were recruited to the PV. Surprisingly, the main ESCRT-III component CHMP4b was massively recruited to the PV, and its expression level was upregulated upon BMDC infection by T. gondii. Finally, we demonstrated that BMDC treatment with U18666A interrupted cholesterol delivery and CHMP4b recruitment to the PV, which interfered with an efficient parasite replication. Altogether, our results highlight the importance of cholesterol trafficking and MVBs formation in DCs for optimal antigen presentation and T. gondii proliferation.

An Arsenic-76 radiotracer to study the routes of assimilation, hemolymph distribution, and tissue inventories in the bioindicator organism Pomacea canaliculata.

The aim of this work was to study the absorption, distribution through the hemolymph, and bioaccumulation of arsenic by the freshwater Pomacea canaliculata using a short-lived tracer (76As, t1/2: 1.07 d) with high specific activity. Arsenic travels mainly dissolved in the plasma of the snail's hemolymph. This element is transferred from the hemolymph to the tissues (87%) 4 h after the inoculation of 50 µL of a 0.04 g/L of 76As radiotracer solution, being the digestive gland, kidney, and head-foot the main places of arsenical inventories. Snails exhibited a rapid arsenic accumulation response in a wide range of concentrations (from 1 to 1000 µg/L) of the metalloid dissolved in water and in a concentration-dependent manner. Also, snails incorporated As from the digestive system when they received a single safe dose of ~2 µg of 76As inoculated in a fish food pellet. The (semi) physiologically based toxicokinetic model developed in this study is based on anatomical and physiological parameters (blood flow, irrigation, tissue volume and other). Together, these findings make P. canaliculata an excellent sentinel organism to evaluate freshwater bodies naturally contaminated with As.

Direct activation of the proton channel by albumin leads to human sperm capacitation and sustained release of inflammatory mediators by neutrophils.

Human voltage-gated proton channels (hHv1) extrude protons from cells to compensate for charge and osmotic imbalances due metabolism, normalizing intracellular pH and regulating protein function. Human albumin (Alb), present at various levels throughout the body, regulates oncotic pressure and transports ligands. Here, we report Alb is required to activate hHv1 in sperm and neutrophils. Dose-response studies reveal the concentration of Alb in semen is too low to activate hHv1 in sperm whereas the higher level in uterine fluid yields proton efflux, allowing capacitation, the acrosomal reaction, and oocyte fertilization. Likewise, Alb activation of hHv1 in neutrophils is required to sustain production and release of reactive oxygen species during the immune respiratory burst. One Alb binds to both voltage sensor domains (VSDs) in hHv1, enhancing open probability and increasing proton current. A computational model of the Alb-hHv1 complex, validated by experiments, identifies two sites in Alb domain II that interact with the VSDs, suggesting an electrostatic gating modification mechanism favoring the active "up" sensor conformation. This report shows how sperm are triggered to fertilize, resolving how hHv1 opens at negative membrane potentials in sperm, and describes a role for Alb in physiology that will operate in the many tissues expressing hHv1.

Epac activation induces an extracellular Ca2+ -independent Ca2+ wave that triggers acrosome reaction in human spermatozoa.

BACKGROUND: The signaling pathways of the intracellular second messengers cAMP and Ca2+ play a crucial role in numerous physiological processes in human spermatozoa. One such process is the acrosome reaction (AR), which is necessary for spermatozoa to traverse the egg envelope and to expose a fusogenic membrane allowing the egg-sperm fusion. Progesterone and zona pellucida elicit an intracellular Ca2+ increase that is needed for the AR in the mammalian spermatozoa. This increase is mediated by an initial Ca2+ influx but also by a release from intracellular Ca2+ stores. It is known that intracellular Ca2+ stores play a central role in the regulation of [Ca2+ ]i and in the generation of complex Ca2+ signals such as oscillations and waves. In the human spermatozoa, it has been proposed that the cAMP analog and specific agonist of Epac 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (2'-O-Me-cAMP) elicits an intracellular Ca2+ release involved in the AR. OBJECTIVE: To identify the molecular entities involved in the Ca2+ mobilization triggered by 2'-O-Me-cAMP in human spermatozoa. MATERIALS AND METHODS: In capacitated human spermatozoa, we monitored Ca2+ dynamics and the occurrence of the AR in real time using Fluo 3-AM and FM4-64 in a Ca2+ -free medium. RESULTS: Epac activation by 2'-O-Me-cAMP induced a Ca2+ wave that started in the midpiece and propagated to the acrosome region. This Ca2+ response was sensitive to rotenone, CGP, xestospongin, NED-19, and thapsigargin, suggesting the participation of different ion transporters (mitochondrial complex I and Na+ /Ca2+ exchanger, inositol 3-phosphate receptors, two-pore channels and internal store Ca2+ -ATPases). DISCUSSION: Our results suggest that Epac activation promotes a dynamic crosstalk between three different intracellular Ca2+ stores: the mitochondria, the redundant nuclear envelope, and the acrosome. CONCLUSION: The Ca2+ wave triggered by Epac activation is necessary to induce the AR and to enhance the flagellar beat.

From cartoons to quantitative models in Golgi transport.

BACKGROUND: Cell biology is evolving to become a more formal and quantitative science. In particular, several mathematical models have been proposed to address Golgi self-organisation and protein and lipid transport. However, most scientific articles about the Golgi apparatus are still using static cartoons that miss the dynamism of this organelle. RESULTS: In this report, we show that schematic drawings of Golgi trafficking can be easily translated into an agent-based model using the Repast platform. The simulations generate an active interplay among cisternae and vesicles rendering quantitative predictions about Golgi stability and transport of soluble and membrane-associated cargoes. The models can incorporate complex networks of molecular interactions and chemical reactions by association with COPASI, a software that handles ordinary differential equations. CONCLUSIONS: The strategy described provides a simple, flexible and multiscale support to analyse Golgi transport. The simulations can be used to address issues directly linked to the mechanism of transport or as a way to incorporate the complexity of trafficking to other cellular processes that occur in dynamic organelles. SIGNIFICANCE: We show that the rules implicitly present in most schematic representations of intracellular trafficking can be used to build dynamic models with quantitative outputs that can be compared with experimental results.

The Synaptotagmin-1 C2B Domain Is a Key Regulator in the Stabilization of the Fusion Pore.

Fusion pores serve as an effective mechanism to connect intracellular organelles and release vesicle contents during exocytosis. A complex lipid rearrangement takes place as membranes approximate, bend, fuse, and establish a traversing water channel to define the fusion pore, linking initially isolated chambers. Thermodynamically, the process is unfavorable and thought to be mediated by specialized proteins. In this work, we have developed a reaction coordinate to induce fusion pores from initially flat and parallel lipid bilayers and we have used it to describe the effects of the synaptotagmin-1 C2B domain during the process. We have obtained free-energy profiles of the whole lipid reorganization in biologically realistic membranes, going from planar and parallel bilayers through stalk hemifusion to water channel formation. Our results point to a lysine-rich polybasic region on synaptotagmin-1 C2B as the key to lipid reorganization control through the formation of phosphatidylinositol bisphosphate clusters that stabilize the fusion pore.

A modelling study highlights the power of detecting and isolating asymptomatic or very mildly affected individuals for COVID-19 epidemic management.

BACKGROUND: Mathematical modelling of infectious diseases is a powerful tool for the design of management policies and a fundamental part of the arsenal currently deployed to deal with the COVID-19 pandemic. METHODS: We present a compartmental model for the disease where symptomatic and asymptomatic individuals move separately. We introduced healthcare burden parameters allowing to infer possible containment and suppression strategies. In addition, the model was scaled up to describe different interconnected areas, giving the possibility to trigger regionalized measures. It was specially adjusted to Mendoza-Argentina's parameters, but is easily adaptable for elsewhere. RESULTS: Overall, the simulations we carried out were notably more effective when mitigation measures were not relaxed in between the suppressive actions. Since asymptomatics or very mildly affected patients are the vast majority, we studied the impact of detecting and isolating them. The removal of asymptomatics from the infectious pool remarkably lowered the effective reproduction number, healthcare burden and overall fatality. Furthermore, different suppression triggers regarding ICU occupancy were attempted. The best scenario was found to be the combination of ICU occupancy triggers (on: 50%, off: 30%) with the detection and isolation of asymptomatic individuals. In the ideal assumption that 45% of the asymptomatics could be detected and isolated, there would be no need for complete lockdown, and Mendoza's healthcare system would not collapse. CONCLUSIONS: Our model and its analysis inform that the detection and isolation of all infected individuals, without leaving aside the asymptomatic group is the key to surpass this pandemic.

Surface charge density and fatty acids enhance the membrane permeation rate of CPP-cargo complexes.

The CPP-effect makes reference to the process by which the membrane translocation rate of a cargo is enhanced by chemical functionalization with cell-penetrating peptides (CPPs). In this work we combine a simple kinetic model with free-energy calculations to explore the energetic basis of the CPP-effect. Two polyglicines are selected as model hydrophilic cargoes, and nona-arginine as a prototypical CPP. We assess the cargo carrying efficiency of nona-arginine by comparing the adsorption and insertion energies of the cargoes, the cargo-free CPPs, and the CPP-cargo complexes, into lipid membranes of varying composition. We also analyze the effect of modifying the type and concentration of anionic lipids, and the implication of these factors on the translocation rate of the CPP-cargo complex. Of particular interest is the evaluation of the catalytic role of palmitic acid (palmitate) as a promoter of the CPP-effect. We also analyse the influence of the size of the cargo on the membrane adsorption and insertion energies. Our results show that the efficiency of nona-arginine as a transmembrane carrier of simple hydrophilic molecules is modulated by the size of the cargo, and is strongly enhanced by increasing the concentration of anionic lipids and of ionized fatty acids in the membrane.

Differential requirement of Rab22a for the recruitment of ER-derived proteins to phagosomes and endosomes in dendritic cells.

The recruitment of endoplasmic reticulum (ER) components to dendritic cell (DC) phagosomes and endosomes is a crucial event to achieve efficient cross-presentation of exogenous antigens. We have previously identified the small GTPase Rab22a as a key regulator of MHC-I trafficking and antigen cross-presentation by DCs. In this study we show that low expression of Rab22a does not prevent the normal delivery of ER-derived proteins to DC phagosomes. In contrast, the presence of these proteins was diminished in endosomes labelled with a fluid phase marker. These observations were confirmed by a functional assay that assesses the translocation of a soluble protein to the cytosol. Interestingly, we also demonstrate that early endosomal maturation is altered in Rab22a deficient DCs. Our results indicate that Rab22a plays a major role in endosomal function and highlight the importance of studying the endocytic and phagocytic pathways separately in DCs.

Intratumor heterogeneity index of breast carcinomas based on DNA methylation profiles.

BACKGROUND: Cancer cells evolve and constitute heterogeneous populations that fluctuate in space and time and are subjected to selection generating intratumor heterogeneity. This phenomenon is determined by the acquisition of genetic/epigenetic alterations and their selection over time which has clinical implications on drug resistance. METHODS: DNA extracted from different tumor cell populations (breast carcinomas, cancer cell lines and cellular clones) were analyzed by MS-MLPA. Methylation profiles were used to generate a heterogeneity index to quantify the magnitude of epigenetic heterogeneity in these populations. Cellular clones were obtained from single cells derived of MDA-MB 231 cancer cell lines applying serial limiting dilution method and morphology was analyzed by optical microscopy and flow cytometry. Clones characteristics were examined through cellular proliferation, migration capacity and apoptosis. Heterogeneity index was also calculated from beta values derived from methylation profiles of TCGA tumors. RESULTS: The study of methylation profiles of 23 fresh breast carcinomas revealed heterogeneous allele populations in these tumor pieces. With the purpose to measure the magnitude of epigenetic heterogeneity, we developed an heterogeneity index based on methylation information and observed that all tumors present their own heterogeneity level. Applying the index calculation in pure cancer cell populations such as cancer cell lines (MDA-MB 231, MCF-7, T47D, HeLa and K-562), we also observed epigenetic heterogeneity. In addition, we detected that clones obtained from the MDA-MB 231 cancer cell line generated their own new heterogeneity over time. Using TCGA tumors, we determined that the heterogeneity index correlated with prognostic and predictive factors like tumor size (p = 0.0088), number of affected axillary nodes (p = 0.007), estrogen receptor expression (p < 0.0001) and HER2 positivity (p = 0.0007). When we analyzed molecular subtypes we found that they presented different heterogeneity levels. Interestingly, we also observed that all mentioned tumor cell populations shared a similar Heterogeneity index (HI) mean. CONCLUSIONS: Our results show that each tumor presents a unique epigenetic heterogeneity level, which is associated with prognostic and predictive factors. We also observe that breast tumor subtypes differ in terms of epigenetic heterogeneity, which could serve as a new contribution to understand the different prognosis of these groups.

Rab22a: A novel regulator of immune functions.

Dendritic cells (DCs) trigger CD8 + T cell responses after the internalization of exogenous antigens in a process called cross-presentation. Multiple intracellular transport events within the endocytic and secretory routes take place in order to accomplish this fundamental immunological process. The endomembrane system can be envisioned as a complex network of membrane domains coordinately working in the fusion of organelles, the budding of vesicles and tubules, and modifying the molecular composition of the limiting membranes. In this context of tightly regulated and dynamic endomembrane transport, small GTPases of the Rab family display a pivotal role by organizing membrane microdomains and defining specific identities to the different intracellular compartments. In this review, we synthesize and update the current knowledge about Rab22a, which has been involved in several immune functions. In this way, we analyze the intracellular localization of Rab22a and its important role in the endocytic recycling, including its relevance during MHC-I trafficking, antigen cross-presentation by DCs and the formation of T cell conjugates. We also describe how different pathogenic microorganisms hijack Rab22a functions to achieve efficient infection and intracellular survival strategies. Furthermore, we examine the oncogenic properties of Rab22a and how its expression determines the progression of many tumors. In summary, we highlight the role of Rab22a as a key effector of the intracellular trafficking that could be exploited in future therapies to modulate the immune system.

A TEM-traceable physiologically functional gold nanoprobe that permeates non-endocytic cells.

BACKGROUND: Nanoparticles' intracellular fate requires proper internalization. Most cells make use of a battery of internalization pathways, but some are practically sealed, as they lack the biochemical machinery for cellular intake. Non-endocytic cells, such as mammals' spermatozoa, challenge standard drug-delivery strategies. PURPOSE: In this article, we present a gold nanoprobe that permeates the external and internal membranes of human sperm. METHODS: Our design makes use of a gold nanoparticle functionalized with a membrane-permeable cysteine-rich recombinant protein. The chimeric protein contains two units of physiologically active metallothioneins (MT) that also provide binding motifs to gold and a cell-penetrating-peptide sequence (CPP) that confers cell permeability to the nanoparticle. RESULTS: Transmission electron microscopy, indirect immunofluorescence, and functional assays show that the nanoprobe is readily internalized in sperm, without compromising cell integrity, while preserving MT's physiological activity. Our findings highlight the potential of CPP-functionalized nanogold for investigating the physiology of otherwise impermeable non-endocytic cells.

Role of human Hv1 channels in sperm capacitation and white blood cell respiratory burst established by a designed peptide inhibitor.

Using a de novo peptide inhibitor, Corza6 (C6), we demonstrate that the human voltage-gated proton channel (hHv1) is the main pathway for H+ efflux that allows capacitation in sperm and permits sustained reactive oxygen species (ROS) production in white blood cells (WBCs). C6 was identified by a phage-display strategy whereby ∼1 million novel peptides were fabricated on an inhibitor cysteine knot (ICK) scaffold and sorting on purified hHv1 protein. Two C6 peptides bind to each dimeric channel, one on the S3-S4 loop of each voltage sensor domain (VSD). Binding is cooperative with an equilibrium affinity (Kd) of ∼1 nM at -50 mV. As expected for a VSD-directed toxin, C6 inhibits by shifting hHv1 activation to more positive voltages, slowing opening and speeding closure, effects that diminish with membrane depolarization.

The interfacial electrostatic potential modulates the insertion of cell-penetrating peptides into lipid bilayers.

Cell-penetrating peptides (CPP) are short sequences of cationic amino-acids that show a surprising ability to traverse lipid bilayers. CPP are considered to be some of the most effective vectors to introduce membrane-impermeable cargos into cells, but the molecular basis of the membrane translocation mechanisms and its dependence on relevant membrane physicochemical properties have yet to be fully determined. In this paper we resort to Molecular Dynamics simulations and experiments to investigate how the electrostatic potential across the lipid/water interface affects the insertion of hydrophilic and amphipathic CPP into two-dimensional lipid structures. Simulations are used to quantify the effect of the transmembrane potential on the free-energy profile associated with the transfer of the CPP across a neutral lipid bilayer. It is found that the electrostatic bias has a relatively small effect on the binding of the peptides to the membrane surface, but that it significantly lowers the permeation barrier. A charge compensation mechanism, arising from the segregation of counter-ions while the peptide traverses the membrane, determines the shape and symmetry of the free-energy curves and underlines relevant mechanistic considerations. Langmuir monolayer experiments performed with a variety of amphiphiles model the incorporation of the CPP into the external membrane leaflet. It is shown that the dipole potential of the monolayer controls the extent of penetration of the CPP into the lipid aggregate, to a greater degree than its surface charge.

Reconstruction of endosomal organization and function by a combination of ODE and agent-based modeling strategies.

BACKGROUND: Reproducing cell processes using an in silico system is an essential tool for understanding the underlying mechanisms and emergent properties of this extraordinary complex biological machine. However, computational models are seldom applied in the field of intracellular trafficking. In a cell, numerous molecular interactions occur on the surface or in the interior of membrane-bound compartments that continually change position and undergo dynamic processes of fusion and fission. At present, the available simulation tools are not suitable to develop models that incorporate the dynamic evolution of the cell organelles. RESULTS: We developed a modeling platform combining Repast (Agent-Based Modeling, ABM) and COPASI (Differential Equations, ODE) that can be used to reproduce complex networks of molecular interactions. These interactions occur in dynamic cell organelles that change position and composition over the course of time. These two modeling strategies are fundamentally different and comprise of complementary capabilities. The ODEs can easily model the networks of molecular interactions, signaling cascades, and complex metabolic reactions. On the other hand, ABM software is especially suited to simulate the movement, interaction, fusion, and fission of dynamic organelles. We used the combined ABM-ODE platform to simulate the transport of soluble and membrane-associated cargoes that move along an endocytic route composed of early, sorting, recycling and late endosomes. We showed that complex processes that strongly depend on transport can be modeled. As an example, the hydrolysis of a GM2-like glycolipid was programmed by adding a trans-Golgi network compartment, lysosomal enzyme trafficking, endosomal acidification, and cholesterol processing to the simulation model. CONCLUSIONS: The model captures the highly dynamic nature of cell compartments that fuse and divide, creating different conditions for each organelle. We expect that this modeling strategy will be useful to understand the logic underlying the organization and function of the endomembrane system. REVIEWERS: This article was reviewed by Drs. Rafael Fernández-Chacón, James Faeder, and Thomas Simmen.

Agents and networks to model the dynamic interactions of intracellular transport.

Cell biology is increasingly evolving to become a more formal and quantitative science. The field of intracellular transport is no exception. However, it is extremely challenging to formulate mathematical and computational models for processes that involve dynamic structures that continuously change their shape, position and composition, leading to information transfer and functional outcomes. The two major strategies employed to represent intracellular trafficking are based on "ordinary differential equations" and "agent-" based modeling. Both approaches have advantages and drawbacks. Combinations of both modeling strategies have promising characteristics to generate meaningful simulations for intracellular transport and allow the formulation of new hypotheses and provide new insights. In the near future, cell biologists will encounter and hopefully overcome the challenge of translating descriptive cartoon representations of biological systems into mathematical network models.