Cell Biological Studies Research Articles

Tumor-associated macrophage-derived GDNF promotes gastric cancer liver metastasis via a GFRA1-modulated autophagy flux

PurposeLiver metastasis, a lethal malignancy of gastric cancer (GC) patients, execrably impairs their prognosis. As yet, however, few studies have been designed to identify the driving molecules during its formation, except screening evidence pausing before their functions or mechanisms. Here, we aimed to survey a key driving event within the invasive margin of liver metastases.MethodsA metastatic GC tissue microarray was used for exploring malignant events during liver-metastasis formation, followed by assessing the expression patterns of glial cell-derived neurotrophic factor (GDNF) and GDNF family receptor alpha 1 (GFRA1). Their oncogenic functions were determined by both loss- and gain-of-function studies in vitro and in vivo, and validated by rescue experiments. Multiple cell biological studies were performed to identify the underlying mechanisms.ResultsIn the invasive margin, GFRA1 was identified as a pivotal molecule involved in cellular survival during liver metastasis formation, and we found that its oncogenic role depends on tumor associated macrophage (TAM)-derived GDNF. In addition, we found that the GDNF-GFRA1 axis protects tumor cells from apoptosis under metabolic stress via regulating lysosomal functions and autophagy flux, and participates in the regulation of cytosolic calcium ion signalling in a RET-independent and non-canonical way.ConclusionFrom our data we conclude that TAMs, homing around metastatic nests, induce the autophagy flux of GC cells and promote the development of liver metastasis via GDNF-GFRA1 signalling. This is expected to improve the comprehension of metastatic pathogenesis and to provide a novel direction of research and translational strategies for the treatment of metastatic GC patients.

Context-specific regulation of extracellular vesicle biogenesis and cargo selection.

To coordinate, adapt and respond to biological signals, cells convey specific messages to other cells. An important aspect of cell-cell communication involves secretion of molecules into the extracellular space. How these molecules are selected for secretion has been a fundamental question in the membrane trafficking field for decades. Recently, extracellular vesicles (EVs) have been recognized as key players in intercellular communication, carrying not only membrane proteins and lipids but also RNAs, cytosolic proteins and other signalling molecules to recipient cells. To communicate the right message, it is essential to sort cargoes into EVs in a regulated and context-specific manner. In recent years, a wealth of lipidomic, proteomic and RNA sequencing studies have revealed that EV cargo composition differs depending upon the donor cell type, metabolic cues and disease states. Analyses of distinct cargo 'fingerprints' have uncovered mechanistic linkages between the activation of specific molecular pathways and cargo sorting. In addition, cell biology studies are beginning to reveal novel biogenesis mechanisms regulated by cellular context. Here, we review context-specific mechanisms of EV biogenesis and cargo sorting, focusing on how cell signalling and cell state influence which cellular components are ultimately targeted to EVs.

Monitoring phosphorylation in proteins using a novel technique: Protein charge transfer spectra

Phosphorylation generates tremendous functional diversity, heterogeneity and complexity of the proteome. Therefore, identifying and understanding phosphorylation is critical in the study of cell biology, disease treatment and prevention. Major limitations related to the conventional techniques used to detect phosphorylation are the usage of high-end instruments, invasive nature and labour-intensive protocols. To overcome these limitations, we employed a simpler, cost-effective and non-invasive technique termed Protein Charge Transfer Spectra (ProCharTS). ProCharTS absorption in near UV-Visible region (250-800 nm) is observed when photoinduced electron transfer occurs from HOMO of electron rich DONOR like Glu/phosphate to the LUMO of electron deficient ACCEPTOR like polypeptide backbone species in the protein matrix. Also, ProCharTS luminescence arises subsequent to ProCharTS absorption when charge recombination of the excited state electron occurs with the created hole. Phosphorylation of a protein introduces extra charge and produces change in its conformation/function. The negatively charged phosphate groups are post translationally attached to the otherwise neutral amino acid which then behaves as anions. Since ProCharTS is sensitive to both the charge content and the proximity among charged residues in a protein, any change in either of them will influence ProCharTS. Moreover, recently it has been reported that the simulated absorption spectra of pSer, pThr and pTyr show charge transfer transitions beyond 200 nm. Here we investigate the ProCharTS of two phosphoproteins, α-Casein and β-Casein under various physiological conditions in both phosphorylated and dephosphorylated states. The phosphorylated proteins were dephosphorylated using Alkaline Phosphatase as confirmed with MALDI and SDS-PAGE. We observed changes in the ProCharTS absorbance and luminescence spectra after dephosphorylation. ProCharTS was found to be sensitive to detect the dephosphorylation in proteins. It can be a potential label-free technique to identify phosphorylation state of a protein.

Aspergillus nidulans.

Aspergillus nidulans is a saprophytic filamentous fungus that is found virtually everywhere. It has a haploid genome, simple nutritional requirements, and rapid growth and reproduction rates. Due to these traits, A. nidulans is a well-established model organism for genetics and cell biology studies, ranging from mitosis, gene discovery and regulation, cell cycle and differentiation, to secondary metabolism. This fungus is extensively used as a tractable and versatile cell factory to yield native and heterologous enzymes, for example, xylanases.

Molecular insights into the effect of alkanediols on FUS liquid-liquid phase separation.

Numerous cell biology studies have used high concentrations of 1,6-hexanediol to dissolve membraneless organelles and disordered protein biomolecular condensates. Yet, little is known about how alkanediols effect liquid-liquid phase separation (LLPS), and why certain alkanediol isomers are more effective. Here, we evaluate the effect of various alkanediols on the archetypal phase separating protein FUS. Low-complexity domain and full-length FUS LLPS is decreased varyingly, while LLPS of FUS RGG-RNA condensates is even enhanced by some alkanediols. NMR experiments show that all diols act similarly, correlating atomistic changes with LLPS-preventing effects. Furthermore, we find no evidence for specific residue interactions - the largest perturbations are seen at backbone and glutamine side-chain hydrogen bonding sites, not hydrophobic/aromatic residues. Furthermore, 1,6 hexanediol favors formation of protein-solvent hydrogen bonds and increases FUS local motions. These findings show how alkanediols affect water-disordered protein interactions, underscoring the difficulty in using alkanediol-derivatives to target dissolution of specific membraneless organelles.

Synchronized Swarmers and Sticky Stalks: Caulobacter crescentus as a Model for Bacterial Cell Biology.

First isolated and classified in the 1960s, Caulobacter crescentus has been instrumental in the study of bacterial cell biology and differentiation. C. crescentus is a Gram-negative alphaproteobacterium that exhibits a dimorphic life cycle composed of two distinct cell types: a motile swarmer cell and a nonmotile, division-competent stalked cell. Progression through the cell cycle is accentuated by tightly controlled biogenesis of appendages, morphological transitions, and distinct localization of developmental regulators. These features as well as the ability to synchronize populations of cells and follow their progression make C. crescentus an ideal model for answering questions relevant to how development and differentiation are achieved at the single-cell level. This review will explore the discovery and development of C. crescentus as a model organism before diving into several key features and discoveries that have made it such a powerful organism to study. Finally, we will summarize a few of the ongoing areas of research that are leveraging knowledge gained over the last century with C. crescentus to highlight its continuing role at the forefront of cell and developmental biology.

Regulation of Tau Expression in Superior Cervical Ganglion (SCG) Neurons In Vivo and In Vitro.

The superior cervical ganglion (SCG) is part of the autonomic nervous system providing sympathetic innervation to the head and neck, and has been regularly used to prepare postnatal neuronal cultures for cell biological studies. We found that during development these neurons change tau expression from the low molecular weight (LMW) isoforms to Big tau, with the potential to affect functions associated with tau such as microtubule dynamic and axonal transport. Big tau contains the large 4a exon that transforms tau from LMW isoforms of 45-60 kDa to 110 kDa. We describe tau expression during postnatal development reporting that the transition from LMW tau to Big tau which started at late embryonic stages is completed by about 4-5 weeks postnatally. We confirmed the presence of Big tau in dissociated postnatal SCG neurons making them an ideal system to study the function of Big tau in neurons. We used SCG explants to examine the response of SCG neurons to lesion and found that Big tau expression returned gradually along the regrowing neurites suggesting that it does not drives regeneration, but facilitates the structure/function of mature SCG neurons. The structural/functional roles of Big tau remain unknown, but it is intriguing that neurons that express Big tau appear less vulnerable to tauopathies.

Xenopus cell-free extracts and their applications in cell biology study.

Xenopus has proven to be a remarkably versatile model organism in the realm of biological research for numerous years, owing to its straightforward maintenance in laboratory settings and its abundant provision of ample-sized oocytes, eggs, and embryos. The cell cycle of these oocytes, eggs, and early embryos exhibits synchrony, and extracts derived from these cells serve various research purposes. Many fundamental concepts in biochemistry, cell biology, and development have been elucidated through the use of cell-free extracts derived from Xenopus cells. Over the past few decades, a wide array of cell-free extracts has been prepared from oocytes, eggs, and early embryos of different Xenopus species at varying cell cycle stages. Each of these extracts possesses distinct characteristics. This review provides a concise overview of the Xenopus species employed in laboratory research, the diverse types of cell-free extracts available, and their respective properties. Furthermore, this review delves into the extensive investigation of spindle assembly in Xenopus egg extracts, underscoring the versatility and potency of these cell-free systems in the realm of cell biology.

Time-Lapse Imaging and Morphometric Analysis of Tracheal Development in Drosophila.

Detailed and quantitative analyses of the cellular events underlying the formation of specific organs or tissues is essential to understand the general mechanisms of morphogenesis and pattern formation. Observation of live tissues or whole-mount fixed specimens has emerged as the method of choice for identifying and quantifying specific cellular and tissular structures within the organism. In both cases, cell and subcellular structure identification and good quality image acquisition for these analyses are essential. Many markers for live imaging and fixed tissue are now available for detecting cell membranes, subcellular structures, and extracellular structures like the extracellular matrix (ECM). Combination of live imaging and analysis of fixed tissue is ideal to obtain a general and detailed picture of the events underlying embryonic development. By applying morphometric methods to both approaches, we can, in addition, obtain a quantitative evaluation of the specific parameters under investigation in morphogenetic and cell biological studies. In this chapter, we focus on the development of the tracheal system of Drosophila melanogaster, which provides an ideal paradigm to understand the formation of branched tubular organs. We describe the most used methods of imaging and morphometric analysis in tubulogenesis using mainly (but not exclusively) examples from embryonic development. We cover embryo preparation for fixed and live analysis of tubulogenesis, together with methods to visualize larval tracheal terminal cell branching and lumen formation. Finally, we describe morphometric analysis and quantification methods using fluorescent images of tracheal cells.

Live Imaging of Nurse Cell Behavior in Late Stages of Drosophila Oogenesis.

Drosophila oogenesis is a powerful and tractable model for studies of cell and developmental biology due to the multitude of well-characterized events in both germline and somatic cells, the ease of genetic manipulation in fruit flies, and the large number of egg chambers produced by each fly. Recent improvements in live imaging and ex vivo culturing protocols have enabled researchers to conduct more detailed, longer-term studies of egg chamber development, enabling insights into fundamental biological processes. Here, we present a protocol for dissection, culturing, and imaging of late-stage egg chambers to study intercellular and directional cytoplasmic flow during "nurse cell dumping." This critical developmental process towards the latter stages of oogenesis (stages 10b/11) results in rapid growth of the oocyte and shrinkage of the nurse cells and is accompanied by dynamic changes in cell shape. We also describe a procedure to record high-time-resolution movies of the flow of unlabeled cytoplasmic contents within nurse cells and through cytoplasmic bridges in the nurse cell cluster using reflection microscopy, and we describe two ways to analyze data from nurse cell dumping.

Generation and delivery of free hydroxyl radicals using a remote plasma

We demonstrate a new gas-based generation source using a low power radio frequency driven atmospheric pressure plasma configured to deliver the radical flux into the far effluent region, well away from interference from other plasma factors such as electric fields, currents, and ultraviolet radiation. Using He–H2O gas chemistry isolated from the laboratory air, the plasma generated flux contains and other radicals including, O and HO2 as well as H2O2 which, along with, was found to vary with H2O vapour content and absorbed power density. Peak flux values were 2.3 nmol s−1 and 0.23 nmol s1 for H2O2 and respectively at a distance of 50 mm from the plasma, with 790 ppmv H2O and a power density of ∼108 W m−3. The maximum flux density was 4.5 × 1019 m−2s−1 falling to 1.7 × 1019 m2 s1 at 110 mm, equivalent to generation rates of 74 µM s1 and 28 µM s−1. Despite high recombination rates at the plasma exit, the escaping flux is still significant, indicating a viable delivery capability to downstream targets. Its performance with regard to generation rates compares well with traditional generation techniques such as radiolysis, advanced oxidation processes and enhanced Fenton-chemistry approaches where production rates are sub-µM s−1. Delivering precisely quantifiable fluxes provides new opportunities for scientific studies and technological opportunities in cell biology, atmospheric chemistry, protein unfolding and systematic dose studies for plasma-based and other related potential medical treatments.

Preparation of Whole-mount Mouse Islets on Vascular Extracellular Matrix for Live Islet Cell Microscopy.

Pancreatic islet β cells preferentially secrete insulin toward the plasma membrane, making contact with the capillary extracellular matrix (ECM). Isolated islets separated from the exocrine acinar cells are the best system for cell biology studies of primary β cells, whereas isolated islets lose their capillary network during ex vivo culture. Providing the appropriate extracellular signaling by attaching islets to vascular ECM-coated surfaces can restore the polarized insulin secretion toward the ECM. The guided secretion toward ECM-coated glass coverslips provides a good model for recording insulin secretion in real time to study its regulation. Additionally, β cells attached to the ECM-coated coverslips are suitable for confocal live imaging of subcellular components including adhesion molecules, cytoskeleton, and ion channels. This procedure is also compatible for total internal reflection fluorescence (TIRF) microscopy, which provides optimal signal-to-noise ratio and high spatial precision of structures close to the plasma membrane. In this article, we describe the optimized protocol for vascular ECM-coating of glass coverslips and the process of attachment of isolated mouse islets on the coverslip. This preparation is compatible with any high-resolution microscopy of live primary β cells. Key features • Optimized coating procedure to attach isolated islets, compatible for both confocal and TIRF microscopy. • The ECM-coated glass coverslip functions as the artificial capillary surface to guide secretion toward the coated surface for optimal imaging of secretion events. • Shows the process of islets attachment to the ECM-coated surface in a 6-day ex vivo culture.

In vitro production of N-degron fused proteins and its application.

The N-degron pathway, first discovered several decades ago by Varshavsky's laboratory, controls the half-life of target proteins depending on their N-terminal residues. In vivo cell biology studies have established the physiological role of the N-degron pathway. However, in vitro studies such as biochemical assays and structural biology studies are relatively limited. The N-degron substrates cannot be obtained via simple protein expression. The N-degron residues are exposed via the proteolytic process from the translated nascent polypeptide chains. Thus, methods for the fusion expression with several cleavable tags and subsequent treatment with specific proteases to design the exposed N-degron signals have been introduced. Recently, we developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B (LC3B), a key marker protein of autophagy, to obtain a high yield of the purified target proteins with variable N-terminal residues for various biochemical studies including enzymatic and binding assays, and crystallization of N-degron complex. This chapter describes the protocols that include the vector map designed for producing LC3B fused target proteins, methods for expression and purification of an example protein, p62/SQSMT1, using different N-terminal residues, and methods to obtain the purified ATG4B protease, which is used for processing LC3B tag and exposing the required N-terminal residues of the target protein.

How Much Do We Know About the Mouse Respiratory Epithelial Stem Cells?

Background-Purpose of this study: Tissue-specific stem cell lines are useful tools for cell biology studies. Information on respiratory tissue cell lines is limited. A doxycycline-regulated epithelial precursor cell line was established from the lung tissue of a tTAxSV40 Tag double transgenic mouse. In this study, we have characterized this cell line in vitro & in vivo, and found to mimic a rare subpopulation of club- and pneumocyte type II-dual cells. Methods: It was partially characterized using cell viability and death assays, H3-thymidine incorporation assay, chloride efflux assay, Western blotting of proteins secreted, RT-PCR assays for RNA isolated. In addition, immune-deficient SCID mice were used as hosts for implantation of this precursor cell line, and feed with/without doxycycline containing water. Immunofluorescent typing using different antibodies were used to characterize the implanted lung. Results: This cell line was found to mimic a rare subpopulation of club- and pneumocyte type II- dual cells with multiple phenotypes. Cell growth was doxycycline-regulated and observed only when doxycycline was omitted from the medium or present at concentrations up to 1 µg/ml, higher concentrations were inhibitory. ACT+ ciliated cells were found upon implantation into immune-deficient mice, in addition. Cell growth was doxycycline-regulated in vitro. When transplanted subcutaneously into immune-deficient mice, these cells migrated to the lung to form organized chimeric structures of donor and host origins, with club cells in the terminal bronchioles, ACT+ ciliated cells along the epithelial lining, and pneumocyte type II-cells in the alveolar interstices. No such homing of donor cells to the lung was observed when the implanted mice were fed doxycycline-containing water. Discussions-Conclusions: This lung stem cell line might be able to provide us with an insight into the differentiation pathway of lung epithelial cells as well as with some understanding of the nature of air trophic-pulmonary epithelial cells. The results of this study underline the possibility of a future application for somatic (stem / precursor) cells in tissue replacement and tissue engineering of the damaged lung. Its ability to secrete and deliver soluble protein, might be a potential novel way for drug delivery. In addition, stem cells are thought to proliferate and differentiate in response to a deficiency or as a result of injury. Successful migration to the target organ and subsequent maturation of these precursors could be attributed to a requirement of lung stem cells to search for an aerated environment. Our findings challenge some current concepts of stem cell biology.This lung stem cell line may become a rich source of cells for tissue engineering and cell-based therapy for lung injury. The route and protocol established for cell introduction into the lung may provide a novel alternative to delivery of soluble protein substances through the airways. This lung stem cell line might also be modified to provide models for screening drugs against respiratory infection.

Chemical Blockage of the Mitochondrial Rhomboid Protease PARL by Novel Ketoamide Inhibitors Reveals Its Role in PINK1/Parkin-Dependent Mitophagy.

The mitochondrial rhomboid protease PARL regulates mitophagy by balancing intramembrane proteolysis of PINK1 and PGAM5. It has been implicated in the pathogenesis of Parkinson's disease, but its investigation as a possible therapeutic target is challenging in this context because genetic deficiency of PARL may result in compensatory mechanisms. To address this problem, we undertook a hitherto unavailable chemical biology strategy. We developed potent PARL-targeting ketoamide inhibitors and investigated the effects of acute PARL suppression on the processing status of PINK1 intermediates and on Parkin activation. This approach revealed that PARL inhibition leads to a robust activation of the PINK1/Parkin pathway without major secondary effects on mitochondrial properties, which demonstrates that the pharmacological blockage of PARL to boost PINK1/Parkin-dependent mitophagy is a feasible approach to examine novel therapeutic strategies for Parkinson's disease. More generally, this study showcases the power of ketoamide inhibitors for cell biological studies of rhomboid proteases.

Comparative Genomics for Evolutionary Cell Biology Using AMOEBAE: Understanding the Golgi and Beyond.

Taking an evolutionary approach to cell biology can yield important new information about how the cell works and how it evolved to do so. This is true of the Golgi apparatus, as it is of all systems within the cell. Comparative genomics is one of the crucial first steps to this line of research, but comes with technical challenges that must be overcome for rigor and robustness. We here introduce AMOEBAE, a workflow for mid-range scale comparative genomic analyses. It allows for customization of parameters, queries, and taxonomic sampling of genomic and transcriptomics data. This protocol article covers the rationale for an evolutionary approach to cell biological study (i.e., when would AMOEBAE be useful), how to use AMOEBAE, and discussion of limitations. It also provides an example dataset, which demonstrates that the Golgi protein AP4 Epsilon is present as the sole retained subunit of the AP4 complex in basidiomycete fungi. AMOEBAE can facilitate comparative genomic studies by balancing reproducibility and speed with user-input and interpretation. It is hoped that AMOEBAE or similar tools will encourage cell biologists to incorporate an evolutionary context into their research.

Proteomics reveals that cell density could affect the efficacy of drug treatment

In vitro cell biology study plays a fundamental role in biological and drug development research, but the repeatability and accuracy of cell studies remain to be low. Various uncertainties during the cell culture process could introduce bias into drug research. In this study, we evaluate the potential effects and underlying mechanisms induced by cell number differences in the cell seeding process. Normally, drug experiments are initiated 24 h after cell seeding, and the difference in the cell number at the time of inoculation leads to the difference in cell confluence (cell density) when drug research is conducted. While cell confluence is closely related to intercellular communication, surface protein interaction, cell autocrine as well as paracrine protein expression of cells, it might have a potential impact on the effect of biological studies such as drug treatment. This study used proteomics technology to comprehensively explore the different protein expression patterns between cells with different confluences. Due to the high sensitivity and high throughput of liquid chromatography-mass spectrometry (LC-MS/MS) detection, it was hired to evaluate the protein expression differences of Hep3B cells with 3 different confluences (30%, 50%, and 70%). The differential expressed proteins were analyzed by the Reactome pathway and the Gene Ontology (GO) pathway. Significant differences were identified across three confluences in terms of the number of proteins identified, the protein expression pattern, and the expression level of certain KEGG pathways. We found that those proteins involved in the cell cycle pathway were differently expressed: the higher the cell confluence, the higher these proteins expressed. A cell cycle inhibitor palbociclib was selected to further verify this observation. Palbociclib in the same dose was applied to cells with different confluence, the results indicated that the growth inhibition effect of palbociclib increases along with the increasing trend of cell cycle protein expression. The result indicated that cell density did influence the effect of drug treatment. Furthermore, three other drugs, cisplatin, paclitaxel, and imatinib, were used to treat the three liver cancer cell lines Hep3B, SUN387, and MHCC97, and a similar observation was obtained that drug effect would be different when the cell confluences were different. Therefore, selecting an appropriate number of cells for plating is vitally important at the beginning of a drug study.

Experience of using acalabrutinib therapy in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma

Introduction. Thanks to scientific advances and discoveries in the study of tumor cell biology, new effective drugs for the treatment of chronic lymphocytic leukemia/ small lymphocytic lymphoma have emerged. Currently, there are drugs with different application points at the molecular level. One such drug is acalabrutinib, which is a selective second-generation inhibitors of Bruton tyrosine kinase and has a more favorable toxicity profile.Objective. To evaluate the efficacy of acalabrutinib in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma.Materials and methods. Since February 2020 acalabrutinib (100 mg 2 p/day orally) has been administered to 7 patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (mean age 64 years) at the Hematology Research Center. Six patients received acalabrutinib in 1st-line therapy and one patient received acalabrutinib in 2nd-line therapy. The mean comorbidity index (CIRS) of the patients was 10 points (range, 8 to 14). Most patients had at least one of the adverse prognostic factors - IGHV nonmutated gene status, TP53 gene aberration (del17p13 and/or TP53 gene mutation), complex karyotype disorders.Results. All patients achieved partial remission of the disease (overall response 100% in the form of regression of B-symptoms, lymphocytic leukocytosis, splenomegaly) at the control period of treatment with acalabrutinib +12 months. The most frequent adverse events when taking acalabrutinib were the occurrence of headache in the first month of taking the drug, spontaneous subcutaneous hematomas. No hematologic toxicity, infectious complications, or cardiac complications were noted. At a median follow-up of 34 months, no patient showed disease progression.Conclusions. The selective Bruton tyrosine kinase inhibitor acalabrutinib has demonstrated high efficacy in patients with adverse risk factors, good tolerability and minimal toxicity, including in comorbid patients.

Microtubule nucleation complex behavior is critical for cortical array homogeneity and xylem wall patterning

Plant cell walls are versatile materials that can adopt a wide range of mechanical properties through controlled deposition of cellulose fibrils. Wall integrity requires a sufficiently homogeneous fibril distribution to cope effectively with wall stresses. Additionally, specific conditions, such as the negative pressure in water transporting xylem vessels, may require more complex wall patterns, e.g., bands in protoxylem. The orientation and patterning of cellulose fibrils are guided by dynamic cortical microtubules. New microtubules are predominantly nucleated from parent microtubules causing positive feedback on local microtubule density with the potential to yield highly inhomogeneous patterns. Inhomogeneity indeed appears in all current cortical array simulations that include microtubule-based nucleation, suggesting that plant cells must possess an as-yet unknown balancing mechanism to prevent it. Here, in a combined simulation and experimental approach, we show that a limited local recruitment of nucleation complexes to microtubules can counter the positive feedback, whereas local tubulin depletion cannot. We observe that nucleation complexes preferentially appear at the plasma membrane near microtubules. By incorporating our experimental findings in stochastic simulations, we find that the spatial behavior of nucleation complexes delicately balances the positive feedback, such that differences in local microtubule dynamics-as in developing protoxylem-can quickly turn a homogeneous array into a banded one. Our results provide insight into how the plant cytoskeleton has evolved to meet diverse mechanical requirements and greatly increase the predictive power of computational cell biology studies.

C-Kit M541L variant is related to ineffective hemopoiesis predisposing to clonal evolution in 3D in vitro biomimetic co-culture model of bone marrow niche

Hematopoietic stem cell (HSC) maintenance in vitro is challenging because stem cell survival relies on cell-to-cell contacts and paracrine signals from bone marrow (BM) microenvironment. Indeed, HSCs easily differentiate in conventional culture systems, and in vitro study of stem cell biology, leukemogenesis, and evolutionary trajectories is limited. 3D-culture systems can mimic tissue architecture and microenvironment thus preserving HSC phenotype. In this study, we developed a calcium alginate hydrogel-based 3D co-culture system of BM mononuclear cells (BMMCs) and BM-derived mesenchymal stem cells (BM-MSCs) to study hemopoiesis in health and disease, such as biological roles of c-Kit M541L somatic mutation of unknown significance. BMMCs and peripheral blood stem cells were obtained from an acute myeloid leukemia patient who experienced graft failure and his haploidentical donor, and from a healthy donor. Cells embedded in alginate scaffolds were cultured for up to 21 days, and flow cytometry immunophenotyping was performed at baseline and every seven days. Our results showed suitability of our 3D culture system in preserving HSC vitality and phenotype throughout the culture period, and also in maintaining composition and vitality of total BMMCs. Moreover, 3D in vitro culture results suggested that M541L c-Kit somatic mutation could be a loss-of-function alteration by reducing HSC maintenance ability thus quickly promoting differentiation, as documented by in vivo graft failure and in vitro absence of long-term culture stability. In conclusions, our 3D BM-like biomimetic culture system allowed long-term stemness maintenance, making it a valid and effective tool for in vitro study of physiological and pathological hemopoiesis.