Biology subjects

Das, A.

Publications and source records attributed to Das, A..

At least 181 records · Page 10Linked to original sources

bioRxiv2022

Conserved basal lamina proteins, laminin and nidogen, are repurposed to organize mechanosensory complexes responsible for touch sensation.

The sense of touch is conferred by the conjoint function of somatosensory neurons and skin cells. These cells meet across a gap filled by a basal lamina, an ancient structure found in metazoans. Using Caenorhabditis elegans, we show that membrane-matrix complexes containing laminin, nidogen, and the MEC-4 mechano-electrical transduction channel reside at this interface and are instrumental for proper touch sensation. These complexes fail to coalesce in touch-insensitive extracellular matrix mutants and in dissociated neurons. MEC-4, but not laminin or nidogen, is destabilized by point mutations in the C-terminal Kunitz domain of the extracellular matrix component, MEC-1. Thus, neuron-epithelial cell interfaces are instrumental in mechanosensory complex assembly and function. Loss of nidogen reduces the density of mechanoreceptor complexes and the amplitude of the touch-evoked currents they carry. These findings imply that somatosensory neurons secrete proteins that actively repurpose the basal lamina to generate special-purpose mechanosensory complexes responsible for vibrotactile sensing.

cell biology↗

bioRxiv2022

The CRISPR-Cas System differentially regulates surface-attached and pellicle-biofilm in Salmonella enterica serovar Typhimurium

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) system has been studied for its role in biofilm regulation and expression of outer membrane proteins in Salmonella. We investigated the CRISPR-Cas mediated biofilm regulation in Salmonella enterica serovar Typhimurium by deleting CRISPR-Cas components,{Delta} crisprI,{Delta} crisprII, {Delta}{Delta}crisprI crisprII, and{Delta} cas op. We determined that the system positively regulates surface-biofilm while inhibiting pellicle-biofilm. In knockout strains, flagellar (fliC, flgK) and curli (csgA) genes were repressed, causing reduced surface-biofilm. Conversely, they displayed altered pellicle-biofilm architecture possessing bacterial multilayers and a denser ECM with enhanced cellulose and lesser Curli, ergo weaker pellicle. The intracellular cellulose concentration was less in the knockout strains due to upregulation of bcsC, necessary for cellulose export. We hypothesized that exported cellulose integrates into the pellicle. We determined that crp is upregulated in the knockout strains, thereby inhibiting the expression of csgD, hence csgA and bcsA. The conflicting upregulation of bcsC, the last gene of bcsABZC operon, could be independently regulated by the CRISPR-Cas system owing to a partial match between the CRISPR-spacers and bcsC gene. The CRP-mediated regulation of the flagellar genes in the knockout strains was probably circumvented through the regulation of Yddx governing the availability of {sigma}28 factor that further regulates class3 flagellar genes (fliC, flgK). Additionally, the variations in the LPS profile and expression of LPS-related genes (rfaC, rfbG, rfbI) in the knockout strains could also contribute to the altered pellicle architecture. Collectively, we establish that the CRISPR-Cas system differentially regulates the formation of surface-attached and pellicle-biofilm.

microbiology↗

bioRxiv2021

BESFA: Bioinformatics based Evolutionary, Structural & Functional Analysis of Prostrate, Placenta, Ovary, Testis, and Embryo (POTE) Paralogs

The POTE family comprises 14 paralogues and is primarily expressed in Prostrate, Placenta, Ovary, Testis, Embryo (POTE), and cancerous cells. The prospective function of the POTE protein family under physiological conditions is less understood. We systematically analyzed their cellular localization and molecular docking analysis to elucidate POTE proteins structure, function, and Adaptive Divergence. Our result discerns that group three POTE paralogs (POTEE, POTEF, POTEI, POTEJ, and POTEKP (a pseudogene)) exhibits significant variation among other members could be because of their Adaptive Divergence. Furthermore, our molecular docking studies on POTE protein revealed the highest binding affinity with NCI-approved anticancer compounds. Additionally, POTEE, POTEF, POTEI, and POTEJ were subject to an explicit molecular dynamic simulation for 50ns. MM-GBSA and other essential electrostatics were calculated that showcased that only POTEE and POTEF have absolute binding affinities with minimum energy exploitation. Thus, this studys outcomes are expected to drive cancer research to successful utilization of POTE genes family as a new biomarker, which could pave the way for the discovery of new therapies.

cancer biology↗

bioRxiv2021

Spontaneous Neuronal Oscillations in the Human Insula are Hierarchically Organized Traveling Waves

The insula plays a fundamental role in a wide range of adaptive human behaviors, but its electrophysiological dynamics are poorly understood. Here we used human intracranial electroencephalographic recordings to investigate the electrophysiological properties and hierarchical organization of spontaneous neuronal oscillations within the insula. We analyzed the neuronal oscillations of the insula directly and found that rhythms in the theta and beta frequency oscillations are widespread and spontaneously present. These oscillations are largely organized along the anterior-posterior axis of the insula. Both the left and right insula showed anterior-to-posterior decreasing gradients for the power of oscillations in the beta frequency band. The left insula also showed a posterior-to-anterior decreasing frequency gradient and an anterior-to-posterior decreasing power gradient in the theta frequency band. In addition to measuring the power of these oscillations, we also examined the phase of these signals across simultaneous recording channels and found that the insula oscillations in the theta and beta bands are traveling waves. The strength of the traveling waves in each frequency was positively correlated with the amplitude of each oscillation. However, the theta and beta traveling waves were uncoupled to each other in terms of phase and amplitude, which suggested that insula traveling waves in the theta and beta bands operate independently. Our findings provide new insights into the spatiotemporal dynamics and hierarchical organization of neuronal oscillations within the insula, which, given its rich connectivity with widespread cortical regions, indicates that oscillations and traveling waves have an important role in intra- and inter-insula communication.

neuroscience↗

bioRxiv2021

Epiphany: predicting Hi-C contact maps from 1D epigenomic signals

Recent deep learning models that predict the Hi-C contact map from DNA sequence achieve promising accuracy but cannot generalize to new cell types and indeed do not capture cell-type-specific differences among training cell types. We propose Epiphany, a neural network to predict cell-type-specific Hi-C contact maps from five epigenomic tracks that are already available in hundreds of cell types and tissues: DNase I hypersensitive sites and ChIP-seq for CTCF, H3K27ac, H3K27me3, and H3K4me3. Epiphany uses 1D convolutional layers to learn local representations from the input tracks, a bidirectional long short-term memory (Bi-LSTM) layers to capture long term dependencies along the epigenome, as well as a generative adversarial network (GAN) architecture to encourage contact map realism. To improve the usability of predicted contact matrices, we trained and evaluated models using multiple normalization and matrix balancing techniques including KR, ICE, and HiC-DC+ Z-score and observed-over-expected count ratio. Epiphany is trained with a combination of MSE and adversarial (i.a., a GAN) loss to enhance its ability to produce realistic Hi-C contact maps for downstream analysis. Epiphany shows robust performance and generalization to held-out chromosomes within and across cell types and species, and its predicted contact matrices yield accurate TAD and significant interaction calls. At inference time, Epiphany can be used to study the contribution of specific epigenomic peaks to 3D architecture and to predict the structural changes caused by perturbations of epigenomic signals.

genomics↗

bioRxiv2021

Synergistic collagen-condiment: Streptococcal collagen-like (Scl) protein in cell-adhesion and diabetic wound-closure matrix

Group A streptococcus (GAS), Streptococcus pyogenes manifests plethora of diseases through its explicit virulence factors. Among these, the recently deciphered MSCRAMMs, Streptococcal collagen-like (Scls) adhesins are most studied proteins in context of their biophysically stable collagenous-sequence (Gly-X-Y) despite the difference from analogous mammalian-collagen. Based on recent evidence on collagen-mimetic Scls, we elucidated biomaterial-potential of the unmodified, recombinant Scl1 (rScl1). Initially, rScl1 trimeric- assembly yielded its stability in silico than the monomeric-unit. Thereby, rScl1 matrix characterization was confirmed in vitro. rScl1 exhibited high A549 and HepG2 cell- viability--rScl1 dose incremented to 20.0 {micro}g/ml at time points up to 24 hr, and on 24 hr stored-dishes--deliberating it non-cytotoxic. Imploring cell-adhesion potential, we observed increased cell-counts tangential to rScl1-gradient. This affirmative prelude on rScl1 as a supporting-matrix cued its synergy to collagen; we discerned it through rScl1-augmented, full-thickness diabetic wound-closure in vivo and as a first, we studied > 18-month rabbit alloxan-models. We have ascertained re-epithelialization with higher type III collagen in absence of inflammation evidenced morphometrically and histologically. Finally, we correlated our observations through atomistic-evaluation of rScl1-2{beta}1-integrin interaction, surprisingly, with augmented binding-energy compared to collagen. Hence, connoting recombinant-streptococcal collagen as an alternate; with further characterization, rScl1 can potentiate important revelations conceding homogeneous and safe, bio-available, biomaterial.

microbiology↗

bioRxiv2021

Active Segregation Dynamics in the Living Cell

In this paper, we bring together our efforts in identifying and understanding nonequilibrium phase segregation driven by active processes in the living cell, with special focus on the segregation of cell membrane components driven by active contractile stresses arising from cortical actomyosin. This also has implications for active segregation dynamics in membraneless regions within the cytoplasm and nucleus (3d). We formulate an active version of the Flory-Huggins theory that incorporates a contribution from fluctuating active stresses. Apart from knitting together some of our past theoretical work in a comprehensive narrative, we highlight some new results, and establish a correspondence with recent studies on Active Model B/B+. We point to the many unusual aspects of the dynamics of active phase segregation, such as (i) anomalous growth dynamics, (ii) coarsening accompanied by propulsion and coalescence of domains that exhibit nonreciprocal effects, (iii) segregation into mesoscale domains, (iv) emergence of a nonequilibrium phase segregated steady state characterised by strong macroscopic fluctuations (fluctuation dominated phase ordering (FDPO)), and (v) mesoscale segregation even above the equilibrium Tc. Apart from its implications for actively driven segregation of binary fluids, these ideas are at the heart of an Active Emulsion description of the lateral organisation of molecules on the plasma membrane of living cells, whose full molecular elaboration appears elsewhere.

biophysics↗

bioRxiv2021

Cells use molecular working memory to navigate in changing chemoattractant fields

In order to migrate over large distances, cells within tissues and organisms rely on sensing local gradient cues. These cues however are multifarious, irregular or conflicting, changing both in time and space. Here we find that single cells utilize a molecular mechanism akin to a working memory, to generate persistent directional migration when signals are disrupted by temporally memorizing their position, while still remaining adaptive to spatial and temporal changes of the signal source. Using dynamical systems theory, we derive that these information processing capabilities are inherent for protein networks whose dynamics is maintained away from steady state through organization at criticality. We demonstrate experimentally using the Epidermal growth factor receptor (EGFR) signaling network, that the memory is maintained in the prolonged receptors activity via a slow-escaping remnant, a dynamical "ghost" of the attractor of the polarized signaling state, that further results in memory in migration. As this state is metastable, it also enables continuous adaptation of the migration direction when the signals vary in space and time. We therefore show that cells implement real-time computations without stable-states to navigate in changing chemoattractant fields by memorizing position of disrupted signals while maintaining sensitivity to novel chemical cues.

biophysics↗

bioRxiv2021

Sketching and sampling approaches for fast and accurate long read classification

BackgroundIn modern sequencing experiments, quickly and accurately identifying the sources of the reads is a crucial need. In metagenomics, where each read comes from one of potentially many members of a community, it can be important to identify the exact species the read is from. In other settings, it is important to distinguish which reads are from the targeted sample and which are from potential contaminants. In both cases, identification of the correct source of a read enables further investigation of relevant reads, while minimizing wasted work. This task is particularly challenging for long reads, which can have a substantial error rate that obscures the origins of each read. ResultsExisting tools for the read classification problem are often alignment or index-based, but such methods can have large time and/or space overheads. In this work, we investigate the effectiveness of several sampling and sketching-based approaches for read classification. In these approaches, a chosen sampling or sketching algorithm is used to generate a reduced representation (a "screen") of potential source genomes for a query readset before reads are streamed in and compared against this screen. Using a query reads similarity to the elements of the screen, the methods predict the source of the read. Such an approach requires limited pre-processing, stores and works with only a subset of the input data, and is able to perform classification with a high degree of accuracy. ConclusionsThe sampling and sketching approaches investigated include uniform sampling, methods based on MinHash and its weighted and order variants, a minimizer-based technique, and a novel clustering-based sketching approach. We demonstrate the effectiveness of these techniques both in identifying the source microbial genomes for reads from a metagenomic long read sequencing experiment, and in distinguishing between long reads from organisms of interest and potential contaminant reads. We then compare these approaches to existing alignment, index and sketching-based tools for read classification, and demonstrate how such a method is a viable alternative for determining the source of query reads. Finally, we present a reference implementation of these approaches at https://github.com/arun96/sketching.

bioinformatics↗

bioRxiv2021

A phylogenetically-restricted essential cell cycle progression factor in the human pathogen Candida albicans

Chromosomal instability in fungal pathogens caused by cell division errors is associated with antifungal drug resistance. To identify mechanisms underlying such instability and to uncover new potential antifungal targets, we conducted an overexpression screen monitoring chromosomal stability in the human fungal pathogen Candida albicans. Analysis of ~1000 genes uncovered six chromosomal stability (CSA) genes, five of which are related to cell division genes in other organisms. The sixth gene, CSA6, is selectively present in the CUG-Ser clade species that includes C. albicans and other human fungal pathogens. The protein encoded by CSA6 localizes to the spindle pole bodies, is required for exit from mitosis, and induces a checkpoint-dependent metaphase arrest upon overexpression. Together, Csa6 defines an essential CUG-Ser fungal clade-specific cell cycle progression factor, highlighting the existence of phylogenetically-restricted cell division genes which may serve as potential unique therapeutic targets. TeaserCsa6 is essential for mitotic progression and mitotic exit in the human fungal pathogen Candida albicans.

cell biology↗

bioRxiv2021

Songbird subthalamic neurons signal song timing and error and project to dopaminergic midbrain

Skill learning requires motor output to be evaluated against internal performance benchmarks. In songbirds, ventral tegmental area (VTA) dopamine neurons (DA) signal performance errors important for learning, but it remains unclear which brain regions project VTA and how these inputs may implement the sensorimotor comparisons necessary for error computation. Here we find that the songbird subthalamic nucleus (STN) projects to VTA and that STN microstimulation can excite VTA neurons. We also discover that STN receives inputs from auditory cortical and ventral pallidal brain regions previously implicated in song evaluation. In the first neural recordings from songbird STN, we discover that the activity of most STN neurons is associated with body movements and not singing, but a small fraction of neurons exhibits precise song timing and performance error signals consistent with performance evaluation. Together our results implicate the STN-VTA projection as an evolutionarily conserved pathway important for motor learning and expand the territories of songbird brain associated with song learning. New & NoteworthySongbird subthalamic (STN) neurons exhibit song-timing and performance error signals and are interconnected with auditory pallium, ventral pallidum and ventral tegmental area, three areas important for song learning.

neuroscience↗

bioRxiv2021

Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity

The E3 ubiquitin ligases CHIP/CHN-1 and UFD-2 team up to accelerate ubiquitin chain formation. However, it remained largely unclear how the high processivity of this E3 set is achieved. Here we studied the molecular mechanism and function of the CHN-1/UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. The HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding and promotes the auto-inhibited CHN-1 state by acting on the conserved position of the U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitinate S-Adenosylhomocysteinase (AHCY-1), an enzyme crucial for lipid metabolism. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation. HIGHLIGHTSO_LIE3 ligase UFD-2 stimulates ubiquitylation activity of CHIP/CHN-1 C_LIO_LIUFD-2 binding promotes dimerization of CHIP/CHN-1 U-box domains and utilization of E2 enzymes C_LIO_LIHSP70/HSP-1 by latching the U-box and TPR domains stabilizes the autoinhibitory state of CHIP/CHN-1, limiting interactions with E2s and UFD-2 C_LIO_LIAssembly with UFD-2 enables CHIP/CHN-1 to regulate lipid metabolism by ubiquitylation of S-Adenosylhomocysteinase C_LI

molecular biology↗

bioRxiv2021

CRISPR-mediated insertion of a chimeric antigen receptor produces nonviral T cell products capable of inducing solid tumor regression

BackgroundChimeric antigen receptor (CAR) T cells traditionally harbor viral vector-based sequences that encode the CAR transgene in the genome. These T cell products have yet to show consistent anti-tumor activity in patients with solid tumors. Further, viral vector manufacturing is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures. MethodsAnti-GD2 CAR T cells were generated using CRISPR/Cas9 within nine days using recombinant Cas9 protein and nucleic acids, without any viral vectors or animal components. The CAR was specifically targeted to the T Cell Receptor Alpha Constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, scRNA-seq, spectral cytometry, and ELISA assays. Functionality was evaluated in vivo in an NSG xenograft neuroblastoma model. ResultsIn comparison to traditional retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory phenotype, and low tonic signaling prior to infusion arising in part from the the knockout of the TCR. Upon exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibited specific cytotoxicity against GD2+ cells in vitro and induced solid tumor regression in vivo, with robust homing, persistence, and low exhaustion against a human neuroblastoma xenograft model. ConclusionsThis proof-of-principle study leveraging virus-free genome editing technology could enable flexible manufacturing of clinically relevant, high-quality CAR T cells to treat cancers, including solid tumors.

bioengineering↗

bioRxiv2021

PICH, an ATP-dependent chromatin remodeling protein, transcriptionally co-regulates oxidative stress response.

Cells respond to oxidative stress by elevating the levels of antioxidants, signaling, and transcriptional regulation often implemented by chromatin remodeling proteins. The study presented in this paper shows that the expression of PICH, an ATP-dependent chromatin remodeler, is upregulated during oxidative stress in HeLa cells. We also show that PICH regulates the expression of Nrf2, a transcription factor regulating antioxidant response, both in the absence and presence of oxidative stress. In turn, Nrf2 regulates the expression of PICH in the presence of oxidative stress. Both PICH and Nrf2 together regulate the expression of antioxidant genes and this transcriptional regulation is dependent on the ATPase activity of PICH. In addition, H3K27ac modification also plays a role in activating transcription in the presence of oxidative stress. Co-immunoprecipitation experiments show that PICH and Nrf2 interact with H3K27ac in the presence of oxidative stress. Mechanistically, PICH recognizes ARE sequences present on its target genes and introduces a conformational change to the DNA sequences leading us to hypothesize that PICH regulates transcription by remodeling DNA. PICH ablation leads to reduced expression of Nrf2 and impaired antioxidant response leading to increased ROS content, thus, showing PICH is essential for the cell to respond to oxidative stress.

molecular biology↗

bioRxiv2021

CD4+ T cell lymphopenia and dysfunction in severe COVID-19 disease is autocrine TNF-α/TNFRI-dependent

Lymphopenia is common in severe COVID-19 disease, yet the mechanisms are poorly understood. In 148 patients with severe COVID-19, we found lymphopenia was associated with worse survival. CD4+ lymphopenia predominated, with lower CD4+/CD8+ ratios in severe COVID-19 compared to recovered, mild disease (p<0.0001). In severe disease, immunodominant CD4+ T cell responses to Spike-1(S1) produced increased in vitro TNF-, but impaired proliferation and increased susceptibility to activation-induced cell death (AICD). CD4+TNF-+ T cell responses inversely correlated with absolute CD4+ counts from severe COVID-19 patients (n=76; R=-0.744, P<0.0001). TNF- blockade including infliximab or anti-TNFRI antibodies strikingly rescued S1-specific CD4+ proliferation and abrogated S1-AICD in severe COVID-19 patients (P<0.001). Single-cell RNAseq demonstrated downregulation of Type-1 cytokines and NF{kappa}B signaling in S1-stimulated CD4+ cells with infliximab treatment. Lung CD4+ T cells in severe COVID-19 were reduced and produced higher TNF- versus PBMC. Together, our findings show COVID-19-associated CD4+ lymphopenia and dysfunction is autocrine TNF-/TNFRI-dependent and therapies targeting TNF- may be beneficial in severe COVID-19. One Sentence SummaryAutocrine TNF-/TNFRI regulates CD4+ T cell lymphopenia and dysfunction in severe COVID-19 disease.

immunology↗

bioRxiv2021

The legacy of maternal SARS-CoV-2 infection on the immunology of the neonate

Despite extensive and ongoing studies of SARS-CoV-2 and evidence that pregnant women are at increased risk of severe COVID-19, the effect of maternal infection on the developing infant remains unclear. To determine the potential impact of exposure to SARS-CoV-2 in utero on the neonate, we have assessed the immunological status of infants born to mothers with confirmed SARS-CoV-2 infection during gestation. No evidence of vertical transmission of SARS-CoV-2 was observed, but transfer of maternal SARS-CoV-2 specific IgG to infants was apparent, although to a lesser extent in cases of active or recent maternal infection. Infants born to mothers with recent/ongoing infection had elevated circulating pro-inflammatory cytokines and enhanced percentages of innate immune cells compared to that seen in infants born to uninfected mothers. In tandem, higher frequencies of FOXP3+ regulatory T cells and circulating IL-10 demonstrated a further nuance to the neonatal effector response. Interestingly, cytokine functionality was enhanced in infants born to mothers exposed to SARS-CoV-2 at any time during pregnancy. This indicates that maternal SARS-CoV-2 infection influences in utero priming of the fetal immune system.

immunology↗

bioRxiv2021

Cellular-resolution monitoring of ischemic stroke pathologies in the rat cortex

Stroke is a leading cause of disability in the Western world. Current post-stroke rehabilitation treatments are only effective in approximately half of the patients. Therefore, there is a pressing clinical need for developing new rehabilitation approaches for enhancing the recovery process, which requires the use of appropriate animal models. Here we study the activity patterns of multiple cortical regions in the rat brain using two-photon microscopy. We longitudinally recorded the fluorescence signal from thousands of neurons labeled with a genetically-encoded calcium indicator before and after an ischemic stroke injury, and found substantial functional changes across motor, somatosensory, and visual cortical regions during the post-stroke cortical reorganization period. We show that a stroke injury in the primary motor cortex has an effect on the activity patterns of neurons not only in the motor and somatosensory cortices, but also in the more distant visual cortex, and that these changes include modified firing rates and kinetics of neuronal activity patterns in response to a sensory stimulus. Changes in neuronal population activity provided animal-specific, circuit-level information on the poststroke cortical reorganization process, which may be essential for evaluating the efficacy of new approaches for enhancing the recovery process.

neuroscience↗

bioRxiv2021

Selection-free non-viral method revealed highly efficient CRISPR-Cas9 genome editing of human pluripotent stem cells guided by cellular autophagy

Cationic liposome-mediated delivery of drugs, DNA, or RNA plays a pivotal role in small molecule therapy, gene editing, and immunization. However, our current knowledge regarding the cellular structures that facilitate this process remains limited. Here, we used human pluripotent stem cells (hPSCs), which form compact colonies consisting of dynamically active cells at the periphery and epithelial-like cells at the core. We discovered that cells at the colony edges selectively got transfected by cationic liposomes through Arp2/3 dependent dynamic lamellipodia, which is augmented by myosin II inhibition. Conversely, cells at the core establish tight junctions at their apical surfaces, impeding liposomal access to the basal lamellipodia and thereby inhibiting transfection. In contrast, liposomes incorporating mannosylated lipids are internalized throughout the entire colony via receptor-mediated endocytosis. These findings contribute a novel mechanistic insight into enhancing therapeutic delivery via liposomes, particularly in cell types characterized by dynamic lamellipodia, such as immune cells, or those comprising the epithelial layer. Significance StatementDrug or gene delivery to human cells is essential for effective treatment. Cationic liposomes provide a safe delivery vehicle compared to viruses. However, the cellular structures required for internalizing liposomes are not yet fully understood. Using human stem cells which grow in compact colonies with more dynamic cells at the periphery and epithelial like cells at the center, here we discovered that Arp2/3 dependent dynamic lamellipodia promotes cationic liposome delivery in dynamic cells while receptor mediated endocytosis is required for epithelial cells. This is significant as it provides mechanisms for enhancing liposome delivery to both migratory and epithelial cells in our body. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/444342v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@800db1org.highwire.dtl.DTLVardef@1031a31org.highwire.dtl.DTLVardef@aca227org.highwire.dtl.DTLVardef@1c9df00_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Mechanisms for liposome transfection to the lamellipodial or epithelial cells. Data shown here suggest cationic liposomes fuse with the negatively charged dynamic lamellipodia membrane in an Arp2/3 dependent manner and the process is enhanced by Myosin II inhibition, such as with the stem cell colony edge cells. However, cells more epithelial in nature such as those inside the stem cell colony center do not possess dynamic lamellipodia at the apical surface, rather they form tight junctions which inhibit cationic liposome transfection. Epithelial cells rely on receptor mediated endocytosis in both Myosin II dependent and independent manners to internalize liposomes with lipids that contain ligands for cell surface receptors such as mannose. C_FIG

cell biology↗