Adenine nucleotide translocase regulates airway epithelial metabolism, surface hydration and ciliary function.

Airway hydration and ciliary function are critical to airway homeostasis and dysregulated in chronic obstructive pulmonary disease (COPD), which is impacted by cigarette smoking and has no therapeutic options. We utilized a high-copy cDNA library genetic selection approach in the amoeba Dictyostelium discoideum to identify genetic protectors to cigarette smoke. Members of the mitochondrial ADP/ATP transporter family adenine nucleotide translocase (ANT) are protective against cigarette smoke in Dictyostelium and human bronchial epithelial cells. Gene expression of ANT2 is reduced in lung tissue from COPD patients and in a mouse smoking model, and overexpression of ANT1 and ANT2 resulted in enhanced oxidative respiration and ATP flux. In addition to the presence of ANT proteins in the mitochondria, they reside at the plasma membrane in airway epithelial cells and regulate airway homeostasis. ANT2 overexpression stimulates airway surface hydration by ATP and maintains ciliary beating after exposure to cigarette smoke, both of which are key functions of the airway. Our study highlights a potential for upregulation of ANT proteins and/or of their agonists in the protection from dysfunctional mitochondrial metabolism, airway hydration and ciliary motility in COPD.This article has an associated First Person interview with the first author of the paper.

4-Hydroxyacetophenone modulates the actomyosin cytoskeleton to reduce metastasis.

Metastases are the cause of the vast majority of cancer deaths. In the metastatic process, cells migrate to the vasculature, intravasate, extravasate, and establish metastatic colonies. This pattern of spread requires the cancer cells to change shape and to navigate tissue barriers. Approaches that block this mechanical program represent new therapeutic avenues. We show that 4-hydroxyacetophenone (4-HAP) inhibits colon cancer cell adhesion, invasion, and migration in vitro and reduces the metastatic burden in an in vivo model of colon cancer metastasis to the liver. Treatment with 4-HAP activates nonmuscle myosin-2C (NM2C) (MYH14) to alter actin organization, inhibiting the mechanical program of metastasis. We identify NM2C as a specific therapeutic target. Pharmacological control of myosin isoforms is a promising approach to address metastatic disease, one that may be readily combined with other therapeutic strategies.

Cancer as a biophysical disease: Targeting the mechanical-adaptability program.

With the number of cancer cases projected to significantly increase over time, researchers are currently exploring "nontraditional" research fields in the pursuit of novel therapeutics. One emerging area that is steadily gathering interest revolves around cellular mechanical machinery. When looking broadly at the physical properties of cancer, it has been debated whether a cancer could be defined as either stiffer or softer across cancer types. With numerous articles supporting both sides, the evidence instead suggests that cancer is not particularly regimented. Instead, cancer is highly adaptable, allowing it to endure the constantly changing microenvironments cancer cells encounter, such as tumor compression and the shear forces in the vascular system and body. What allows cancer cells to achieve this adaptability are the particular proteins that make up the mechanical network, leading to a particular mechanical program of the cancer cell. Coincidentally, some of these proteins, such as myosin II, α-actinins, filamins, and actin, have either altered expression in cancer and/or some type of direct involvement in cancer progression. For this reason, targeting the mechanical system as a therapeutic strategy may lead to more efficacious treatments in the future. However, targeting the mechanical program is far from trivial. As involved as the mechanical program is in cancer development and metastasis, it also helps drive many other key cellular processes, such as cell division, cell adhesion, metabolism, and motility. Therefore, anti-cancer treatments targeting the mechanical program must take great care to avoid potential side effects. Here, we introduce the potential of targeting the mechanical program while also providing its challenges and shortcomings as a strategy for cancer treatment.

Particle-based model of mechanosensory contractility kit assembly.

Cell shape change processes, such as proliferation, polarization, migration, and cancer metastasis, rely on a dynamic network of macromolecules. The proper function of this network enables mechanosensation, the ability of cells to sense and respond to mechanical cues. Myosin II and cortexillin I, critical elements of the cellular mechanosensory machinery, preassemble in the cytoplasm of Dictyostelium cells into complexes that we have termed contractility kits (CKs). Two IQGAP proteins then differentially regulate the mechanoresponsiveness of the cortexillin I-myosin II elements within CKs. To investigate the mechanism of CK self-assembly and gain insight into possible molecular means for IQGAP regulation, we developed a coarse-grained excluded volume molecular model in which all protein polymers are represented by nm-sized spheres connected by spring-like links. The model is parameterized using experimentally measured parameters acquired through fluorescence cross-correlation spectroscopy and fluorescence correlation spectroscopy, which describe the interaction affinities and diffusion coefficients for individual molecular components, and which have also been validated via several orthogonal methods. Simulations of wild-type and null-mutant conditions implied that the temporal order of assembly of these kits is dominated by myosin II dimer formation and that IQGAP proteins mediate cluster growth. In addition, our simulations predicted the existence of "ambiguous" CKs that incorporate both classes of IQGAPs, and we confirmed this experimentally using fluorescence cross-correlation spectroscopy. The model serves to describe the formation of the CKs and how their assembly enables and regulates mechanosensation at the molecular level.

A mesoscale mechanical model of cellular interactions.

Computational models of cell mechanics allow the precise interrogation of cell shape change. These morphological changes are required for cells to survive in diverse tissue environments. Here, we present a mesoscale mechanical model of cell-substrate interactions using the level set method based on experimentally measured parameters. By implementing a viscoelastic mechanical equivalent circuit, we accurately model whole-cell deformations that are important for a variety of cellular processes. To effectively model shape changes as a cell interacts with a substrate, we have included receptor-mediated adhesion, which is governed by catch-slip bond behavior. The effect of adhesion was explored by subjecting cells to a variety of different substrates including flat, curved, and deformable surfaces. Finally, we increased the accuracy of our simulations by including a deformable nucleus in our cells. This model sets the foundation for further exploration into computational analyses of multicellular interactions.

The mechanobiome: a goldmine for cancer therapeutics.

Cancer progression is dependent on heightened mechanical adaptation, both for the cells' ability to change shape and to interact with varying mechanical environments. This type of adaptation is dependent on mechanoresponsive proteins that sense and respond to mechanical stress, as well as their regulators. Mechanoresponsive proteins are part of the mechanobiome, which is the larger network that constitutes the cell's mechanical systems that are also highly integrated with many other cellular systems, such as gene expression, metabolism, and signaling. Despite the altered expression patterns of key mechanobiome proteins across many different cancer types, pharmaceutical targeting of these proteins has been overlooked. Here, we review the biochemistry of key mechanoresponsive proteins, specifically nonmuscle myosin II, α-actinins, and filamins, as well as the partnering proteins 14-3-3 and CLP36. We also examined a wide range of data sets to assess how gene and protein expression levels of these proteins are altered across many different cancer types. Finally, we determined the potential of targeting these proteins to mitigate invasion or metastasis and suggest that the mechanobiome is a goldmine of opportunity for anticancer drug discovery and development.

Why new biology must be uncovered to advance therapeutic strategies for chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is characterized by the destruction of alveolar tissue (in emphysema) and airway remodeling (leading to chronic bronchitis), which cause difficulties in breathing. It is a growing public health concern with few therapeutic options that can reverse disease progression or mortality. This is in part because current treatments mainly focus on ameliorating symptoms induced by inflammatory pathways as opposed to curing disease. Hence, emerging research focused on upstream pathways are likely to be beneficial in the development of efficient therapeutics to address the root causes of disease. Some of these pathways include mitochondrial function, cytoskeletal structure and maintenance, and airway hydration, which are all affected by toxins that contribute to COPD. Because of the complexity of COPD and unknown targets for disease onset, simpler model organisms have proved to be useful tools in identifying disease-relevant pathways and targets. This review summarizes COPD pathology, current treatments, and therapeutic discovery research, with a focus on the aforementioned pathways that can advance the therapeutic landscape of COPD.

How the mechanobiome drives cell behavior, viewed through the lens of control theory.

Cells have evolved sophisticated systems that integrate internal and external inputs to coordinate cell shape changes during processes, such as development, cell identity determination, and cell and tissue homeostasis. Cellular shape-change events are driven by the mechanobiome, the network of macromolecules that allows cells to generate, sense and respond to externally imposed and internally generated forces. Together, these components build the cellular contractility network, which is governed by a control system. Proteins, such as non-muscle myosin II, function as both sensors and actuators, which then link to scaffolding proteins, transcription factors and metabolic proteins to create feedback loops that generate the foundational mechanical properties of the cell and modulate cellular behaviors. In this Review, we highlight proteins that establish and maintain the setpoint, or baseline, for the control system and explore the feedback loops that integrate different cellular processes with cell mechanics. Uncovering the genetic, biophysical and biochemical interactions between these molecular components allows us to apply concepts from control theory to provide a systems-level understanding of cellular processes. Importantly, the actomyosin network has emerged as more than simply a 'downstream' effector of linear signaling pathways. Instead, it is also a significant driver of cellular processes traditionally considered to be 'upstream'.

Contractility kits promote assembly of the mechanoresponsive cytoskeletal network.

Cellular contractility is governed by a control system of proteins that integrates internal and external cues to drive diverse shape change processes. This contractility controller includes myosin II motors, actin crosslinkers and protein scaffolds, which exhibit robust and cooperative mechanoaccumulation. However, the biochemical interactions and feedback mechanisms that drive the controller remain unknown. Here, we use a proteomics approach to identify direct interactors of two key nodes of the contractility controller in the social amoeba Dictyostelium discoideum: the actin crosslinker cortexillin I and the scaffolding protein IQGAP2. We highlight several unexpected proteins that suggest feedback from metabolic and RNA-binding proteins on the contractility controller. Quantitative in vivo biochemical measurements reveal direct interactions between myosin II and cortexillin I, which form the core mechanosensor. Furthermore, IQGAP1 negatively regulates mechanoresponsiveness by competing with IQGAP2 for binding the myosin II-cortexillin I complex. These myosin II-cortexillin I-IQGAP2 complexes are pre-assembled into higher-order mechanoresponsive contractility kits (MCKs) that are poised to integrate into the cortex upon diffusional encounter coincident with mechanical inputs.This article has an associated First Person interview with the first author of the paper.

Helping Scholars Overcome Socioeconomic Barriers to Medical and Biomedical Careers: Creating a Pipeline Initiative.

Problem: To achieve their potential in medical and biomedical careers, students (scholars) from under-resourced backgrounds must build sophisticated skills and develop confidence and professionalism. To flourish in an advanced educational system that may be unfamiliar, these scholars also need networks of mentors and role models. These challenges can affect scholars at multiple stages of their education. Intervention: To meet these challenges, we created a broad and innovative biomedical research-focused pipeline program: the Johns Hopkins Initiative for Careers in Science in Medicine (CSM Initiative). This initiative targets three levels: high school, undergraduate, and post-baccalaureate/pre-doctoral (graduate and medical). We provide training in essential academic, research, professional, and social skills to meet the unique challenges of our scholars from under-resourced backgrounds. Scholars also build relationships with mentors who provide career guidance and support. We present an overview of the training and assessment at each level of this initiative. Context: The initiative took place at an institution located in the greater Baltimore area and that is endowed with exceptional doctoral and postdoctoral trainees, staff, and faculty including clinicians, physician-scientists, and scientists who served as key role models and mentors. Our pipeline program draws from local high school students and a local and national pool of undergraduates and post-baccalaureates preparing for medical or graduate school. Impact: Our goals for the high school scholars are significant improvement in academic skills, increased confidence, and matriculation into higher education systems. Currently, at least 83% of high school scholars have matriculated into four-year college programs and 73% have chosen science, technology, engineering, math, and medicine (STEMM)-related majors. Among undergraduate participants, 42% have matriculated thus far into medical or biomedical graduate programs and this number is expected to rise as more scholars graduate from college and either enter graduate training or pursue STEMM careers. Another 25% have returned to our post-baccalaureate program. Among post-baccalaureate scholars, 71% have now matriculated into doctoral-level graduate biomedical programs (medical or graduate school) and the remaining 29% are pursuing careers in STEMM-related fields such as biomedical research with some still aiming at graduate-level education. Our long-term goal is to see a large majority of our scholars become successful professionals in medicine, biomedical research, allied healthcare, or other STEMM fields. Analysis of the early phases of the CSM initiative demonstrates such outcomes are attainable. Lessons Learned: This program provides experiences in which scholars develop and practice core competencies essential for developing their self-identity as scientists and professionals. The most important lesson learned is that mentorship teams must be highly dynamic, flexible, thoughtful, and personal in responding to the wide range of challenges and obstacles that scholars from under-resourced backgrounds must overcome to achieve career success.

The fifth sense: Mechanosensory regulation of alpha-actinin-4 and its relevance for cancer metastasis.

Metastatic cancer cells invading through dense tumor stroma experience internal and external forces that are sensed through a variety of mechanosensory proteins that drive adaptations for specific environments. Alpha-actinin-4 (ACTN4) is a member of the α-actinin family of actin crosslinking proteins that is upregulated in several types of cancers. It shares 86% protein similarity with α-actinin-1, another non-muscle ACTN isoform, which appears to have a more modest role, if any, in cancer progression. While they share regulatory mechanisms, such as phosphorylation, calcium binding, phosphatidyl inositol binding, and calpain cleavage, α-actinin-4 exhibits a unique mechanosensory regulation that α-actinin-1 does not. This behavior is mediated, at least in part, by each protein's actin-binding affinity as well as the catch-slip-bond behavior of the actin binding domains. We will discuss currently known modes of ACTN4 regulation, their interactions, and how mechanosensation may provide major therapeutic targeting potential for cancer metastasis.

14-3-3 proteins tune non-muscle myosin II assembly.

The 14-3-3 family comprises a group of small proteins that are essential, ubiquitous, and highly conserved across eukaryotes. Overexpression of the 14-3-3 proteins σ, ϵ, ζ, and η correlates with high metastatic potential in multiple cancer types. In Dictyostelium, 14-3-3 promotes myosin II turnover in the cell cortex and modulates cortical tension, cell shape, and cytokinesis. In light of the important roles of 14-3-3 proteins across a broad range of eukaryotic species, we sought to determine how 14-3-3 proteins interact with myosin II. Here, conducting in vitro and in vivo studies of both Dictyostelium (one 14-3-3 and one myosin II) and human proteins (seven 14-3-3s and three nonmuscle myosin IIs), we investigated the mechanism by which 14-3-3 proteins regulate myosin II assembly. Using in vitro assembly assays with purified myosin II tail fragments and 14-3-3, we demonstrate that this interaction is direct and phosphorylation-independent. All seven human 14-3-3 proteins also altered assembly of at least one paralog of myosin II. Our findings indicate a mechanism of myosin II assembly regulation that is mechanistically conserved across a billion years of evolution from amebas to humans. We predict that altered 14-3-3 expression in humans inhibits the tumor suppressor myosin II, contributing to the changes in cell mechanics observed in many metastatic cancers.

Cytokinesis: Robust cell shape regulation.

Cytokinesis, the final step of cell division, is a great example of robust cell shape regulation. A wide variety of cells ranging from the unicellular Dictyostelium to human cells in tissues proceed through highly similar, stereotypical cell shape changes during cell division. Typically, cells first round up forming a cleavage furrow in the middle, which constricts resulting in the formation of two daughter cells. Tight control of cytokinesis is essential for proper segregation of genetic and cellular materials, and its failure is deleterious to cell viability. Thus, biological systems have developed elaborate mechanisms to ensure high fidelity of cytokinesis, including the existence of multiple biochemical and mechanical pathways regulated through feedback. In this review, we focus on the built-in redundancy of the cytoskeletal machinery that allows cells to divide successfully in a variety of biological and mechanical contexts. Using Dictyostelium cytokinesis as an example, we demonstrate that the crosstalk between biochemical and mechanical signaling through feedback ensures correct assembly and function of the cell division machinery.

Pharmacological activation of myosin II paralogs to correct cell mechanics defects.

Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human myosin IIB (MYH10) and human myosin IIC (MYH14), but not human myosin IIA (MYH9). We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof of concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior.

Mechanochemical Signaling Directs Cell-Shape Change.

For specialized cell function, as well as active cell behaviors such as division, migration, and tissue development, cells must undergo dynamic changes in shape. To complete these processes, cells integrate chemical and mechanical signals to direct force production. This mechanochemical integration allows for the rapid production and adaptation of leading-edge machinery in migrating cells, the invasion of one cell into another during cell-cell fusion, and the force-feedback loops that ensure robust cytokinesis. A quantitative understanding of cell mechanics coupled with protein dynamics has allowed us to account for furrow ingression during cytokinesis, a model cell-shape-change process. At the core of cell-shape changes is the ability of the cell's machinery to sense mechanical forces and tune the force-generating machinery as needed. Force-sensitive cytoskeletal proteins, including myosin II motors and actin cross-linkers such as α-actinin and filamin, accumulate in response to internally generated and externally imposed mechanical stresses, endowing the cell with the ability to discern and respond to mechanical cues. The physical theory behind how these proteins display mechanosensitive accumulation has allowed us to predict paralog-specific behaviors of different cross-linking proteins and identify a zone of optimal actin-binding affinity that allows for mechanical stress-induced protein accumulation. These molecular mechanisms coupled with the mechanical feedback systems ensure robust shape changes, but if they go awry, they are poised to promote disease states such as cancer cell metastasis and loss of tissue integrity.

Cigarette smoke disrupts monolayer integrity by altering epithelial cell-cell adhesion and cortical tension.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality. Cigarette smoke (CS) drives disease development and progression. The epithelial barrier is damaged by CS with increased monolayer permeability. However, the molecular changes that cause this barrier disruption and the interaction between adhesion proteins and the cytoskeleton are not well defined. We hypothesized that CS alters monolayer integrity by increasing cell contractility and decreasing cell adhesion in epithelia. Normal human airway epithelial cells and primary COPD epithelial cells were exposed to air or CS, and changes measured in protein levels. We measured the cortical tension of individual cells and the stiffness of cells in a monolayer. We confirmed that the changes in acute and subacute in vitro smoke exposure reflect protein changes seen in cell monolayers and tissue sections from COPD patients. Epithelial cells exposed to repetitive CS and those derived from COPD patients have increased monolayer permeability. E-cadherin and ß-catenin were reduced in smoke exposed cells as well as in lung tissue sections from patients with COPD. Moreover, repetitive CS caused increased tension in individual cells and cells in a monolayer, which corresponded with increased polymerized actin without changes in myosin IIA and IIB total abundance. Repetitive CS exposure impacts the adhesive intercellular junctions and the tension of epithelial cells by increased actin polymer levels, to further destabilize cell adhesion. Similar changes are seen in epithelial cells from COPD patients indicating that these findings likely contribute to COPD pathology.

Yes-associated protein impacts adherens junction assembly through regulating actin cytoskeleton organization.

The Hippo pathway effector Yes-associated protein (YAP) regulates liver size by promoting cell proliferation and inhibiting apoptosis. However, recent in vivo studies suggest that YAP has important cellular functions other than controlling proliferation and apoptosis. Transgenic YAP expression in mouse hepatocytes results in severe jaundice. A possible explanation for the jaundice could be defects in adherens junctions that prevent bile from leaking into the blood stream. Indeed, immunostaining of E-cadherin and electron microscopic examination of bile canaliculi of Yap transgenic livers revealed abnormal adherens junction structures. Using primary hepatocytes from Yap transgenic livers and Yap knockout livers, we found that YAP antagonizes E-cadherin-mediated cell-cell junction assembly by regulating the cellular actin architecture, including its mechanical properties (elasticity and cortical tension). Mechanistically, we found that YAP promoted contractile actin structure formation by upregulating nonmuscle myosin light chain expression and cellular ATP generation. Thus, by modulating actomyosin organization, YAP may influence many actomyosin-dependent cellular characteristics, including adhesion, membrane protrusion, spreading, morphology, and cortical tension and elasticity, which in turn determine cell differentiation and tissue morphogenesis.

Cortical mechanics and myosin-II abnormalities associated with post-ovulatory aging: implications for functional defects in aged eggs.

STUDY HYPOTHESIS: Cellular aging of the egg following ovulation, also known as post-ovulatory aging, is associated with aberrant cortical mechanics and actomyosin cytoskeleton functions. STUDY FINDING: Post-ovulatory aging is associated with dysfunction of non-muscle myosin-II, and pharmacologically induced myosin-II dysfunction produces some of the same deficiencies observed in aged eggs. WHAT IS KNOWN ALREADY: Reproductive success is reduced with delayed fertilization and when copulation or insemination occurs at increased times after ovulation. Post-ovulatory aged eggs have several abnormalities in the plasma membrane and cortex, including reduced egg membrane receptivity to sperm, aberrant sperm-induced cortical remodeling and formation of fertilization cones at the site of sperm entry, and reduced ability to establish a membrane block to prevent polyspermic fertilization. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Ovulated mouse eggs were collected at 21-22 h post-human chorionic gonadotrophin (hCG) (aged eggs) or at 13-14 h post-hCG (young eggs), or young eggs were treated with the myosin light chain kinase (MLCK) inhibitor ML-7, to test the hypothesis that disruption of myosin-II function could mimic some of the effects of post-ovulatory aging. Eggs were subjected to various analyses. Cytoskeletal proteins in eggs and parthenogenesis were assessed using fluorescence microscopy, with further analysis of cytoskeletal proteins in immunoblotting experiments. Cortical tension was measured through micropipette aspiration assays. Egg membrane receptivity to sperm was assessed in in vitro fertilization (IVF) assays. Membrane topography was examined by low-vacuum scanning electron microscopy (SEM). MAIN RESULTS AND THE ROLE OF CHANCE: Aged eggs have decreased levels and abnormal localizations of phosphorylated myosin-II regulatory light chain (pMRLC; P = 0.0062). Cortical tension, which is mediated in part by myosin-II, is reduced in aged mouse eggs when compared with young eggs, by ∼40% in the cortical region where the metaphase II spindle is sequestered and by ∼50% in the domain to which sperm bind and fuse (P < 0.0001). Aging-associated parthenogenesis is partly rescued by treating eggs with a zinc ionophore (P = 0.003), as is parthenogenesis induced by inhibition of mitogen-activated kinase (MAPK) 3/1 [also known as extracellular signal-regulated kinase (ERK)1/2] or MLCK. Inhibition of MLCK with ML-7 also results in effects that mimic those of post-ovulatory aging: fertilized ML-7-treated eggs show both impaired fertilization and increased extents of polyspermy, and ML-7-treated young eggs have several membrane abnormalities that are shared by post-ovulatory aged eggs. LIMITATIONS, REASONS FOR CAUTION: These studies were done with mouse oocytes, and it remains to be fully determined how these findings from mouse oocytes would compare with other species. For studies using methods not amenable to analysis of large sample sizes and data are limited to what images one can capture (e.g. SEM), data should be interpreted conservatively. WIDER IMPLICATIONS OF THE FINDINGS: These data provide insights into causes of reproductive failures at later post-copulatory times. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: This project was supported by R01 HD037696 and R01 HD045671 from the NIH to J.P.E. Cortical tension studies were supported by R01 GM66817 to D.N.R. The authors declare there are no financial conflicts of interest.

An RNA-binding protein, RNP-1, protects microtubules from nocodazole and localizes to the leading edge during cytokinesis and cell migration in Dictyostelium cells.

AIM: RNA-binding proteins are a large group of regulators (800-1000 in humans), some of which play significant roles in mRNA local translation. In this study, we analyzed the functions of the protein RNP-1, which was previously discovered in a genetic selection screen for nocodazole suppression. METHODS: The growth rates and the microtubule networks of Dictyostelium cells were assessed with or without nocodazole (10 µmol/L) in suspension culture. Fluorescent images of RNP-1-GFP and RFP-tubulin were captured when cells were undergoing cytokinesis, then the GFP signal intensity and distance to the nearest centrosome were analyzed by using a computer program written in Matlab®. The RNP-1-GFP-expresseding cells were polarized, and the time-lapse images of cells were captured when cells were chemotaxing to a cAMP source. RESULTS: Over-expression of RNP-1 rescued the growth defects caused by the microtubule-destabilizing agent nocodazole. Over-expression of RNP-1 protected microtubules from nocodazole treatment. In cells undergoing cytokinesis, the RNP-1 protein was localized to the polar regions of the cell cortex, and protein levels decreased proportionally as the power of the distance from the cell cortex to the nearest centrosome. In chemotactic cells, the RNP-1 protein localized to the leading edge of moving cells. Sequence analysis revealed that RNP-1 has two RNA-binding domains and is related to cytosolic poly(A)-binding proteins (PABPCs) in humans. CONCLUSION: RNP-1 has roles in protecting microtubules and in directing cortical movement during cytokinesis and cell migration in Dictyostelium cells. The sequence similarity of RNP-1 to human PABPCs suggests that PABPCs may have similar functions in mammalian cells, perhaps in regulating microtubule dynamics and functions during cortical movement in cytokinesis and cell migration.