Advances in gene therapy increasingly depend on understanding how viral vectors behave within complex, multilayered human tissues. While retinal organoids serve as a powerful model for studying AAV efficacy, their dense, light-scattering architecture has historically limited the ability to visualize and quantify transduction at single-cell resolution. Conventional nuclear stains suffer from rapid photobleaching, cytotoxicity, and shallow imaging depth which hinder repeated live imaging and prevent accurate 3D cell segmentation throughout the organoid. Conventional membrane dyes also pose challenges for staining organoids due to poor penetration, uneven labeling, and rapid internalization by endocytosis.
In a 2025 Small Methods publication, Rogler et. al. developed a longitudinal imaging and deep-learning pipeline to map single-cell AAV transduction dynamics in intact human retinal organoids. This approach required robust and photostable live-cell stains compatible with deep (>100 µm) confocal imaging and repeated imaging over many days. To meet this need, the authors selected Biotium’s far-red NucSpot® Live 650 Nuclear Stain, which provides bright, uniform labeling with minimal phototoxicity and exceptional light penetration compared to blue- or green-excitable DNA dyes. CellBrite® Steady 550, a unique stain for long-term labeling of membranes in live cells, was also used for manual quantification of transduced cells to gauge the performance of their deep-learning method.
Using NucSpot® Live 650, the team captured high-contrast 3D nuclear signals across entire organoids and enabled the use of Cellpose, a deep-learning segmentation algorithm. Paired with GFP-expressing AAV reporters, this allowed precise quantification of transduced cells, as well as quantification of how transduction patterns evolve over time and spatial depth.
The end result revealed heterogeneous AAV penetration profiles, cell-type-specific susceptibility, and spatial gradients of transduction that would have been obscured using conventional methods. Biotium’s NucSpot® Live 650 Nuclear Stain and CellBrite® Steady 550 Membrane Stain enabled high-fidelity, longitudinal imaging in thick living tissues, making quantitative AAV mapping in 3D retinal models possible.
Confocal image of the center plane of the 3D stack of a 264 days old human retinal organoid without virus, stained with NucSpot Live 650 (magenta) and CellBrite Steady 550 (white). Credit: Rogler et al., Small Methods (2025). Reproduced under CC BY 4.0.
Rogler, T. S., Salbaum, K. A., Brinkop, A. T., Sonntag, S. M., James, R., Shelton, E. R., Thielen, A., Rose, R., Babutzka, S., Klopstock, T., Michalakis, S., & Serwane, F. (2025). 3D quantification of viral transduction efficiency in living human retinal organoids. Small Methods, 2025 Jun 12, e2401050. https://doi.org/10.1002/smtd.202401050
In membrane biology, the diffusion and interaction of proteins and lipids are fundamental to cellular signaling and function. Neurotransmitter receptors such as the nicotinic acetylcholine receptor (nAChR) are known to display cholesterol-sensitive diffusion patterns in the plasma membrane, but conventional microscopy has lacked the spatiotemporal resolution to resolve these dynamics. Capturing rapid, heterogeneous motions requires Minimal Photon Flux (MINFLUX), a super-resolution microscopy method that merges Stimulated Emission Depletion (STED) and single-molecule localization (SMLM) to deliver nanometer precision with sub-millisecond resolution.
In a 2025 Nature Communicationspublication, Reina et al. applied two-color MINFLUX single-molecule tracking to visualize the joint motion of nAChRs and fluorescent cholesterol in live mammalian cells. To enable this approach, the authors used Biotium’s CF®640– and CF®680R-labeled α-bungarotoxin conjugates to selectively tag nAChRs. CF®680R Dye provided the critical spectral separation needed for multiplex detection of a cholesterol analogue labeled with STAR Red dye. This allowed the researchers to reliably co-track receptor and lipid molecules under single-wavelength excitation, revealing heterogeneous diffusion modes and direct instances of receptor–cholesterol co-diffusion.
The authors also showed that cholesterol availability modulates receptor motion, with distinct regions of confined and joint diffusion observed. These findings, made possible by Biotium’s wide selection of CF® Bungarotoxin conjugates, underscore cholesterol’s role in shaping receptor dynamics and highlight how high-resolution, multiplexed MINFLUX microscopy can uncover lipid–protein interactions previously imperceptible with conventional methods.
Representative data of detector channel ratio (DCR) and intensity values (as effective counts at offset (ECO)) over time steps (upper two panels) and corresponding tracks in the X-Y plane with colours indicating classification as fPEG-Chol (orange), CF®680R-BTX (blue), and co-diffusion (black) for cells with fPEG-Chol alone, CF®680R-BTX nAChR alone, and the 2-colour co-labelling experiments with both fPEG-Chol and CF®680R-BTX. Co-diffusion becomes apparent in the increased ECO values and simultaneous change in DCR values converging in a corridor between 0.40 and 0.55 (marked by the two horizontal dashed lines). Scale bar = 100 nm. Modified from Reina et. al., Nat. Comm., 16 (2025). Reproduced under CC BY 4.0.MINFLUX single-molecule tracking of individual bungarotoxin-labelled nicotinic acetylcholine receptors and fluorescent cholesterol in live cells, ensembled-averaged (EA), time-averaged (TA) mean squared displacement (EA-TA-MSD) and TA-MSD analyses, 2D-diffusion parameters and diffusive regimes derived from the anomalous coefficient β. Credit: Reina et. al., Nat. Comm., 16 (2025). Reproduced under CC BY 4.0.
Reina, F., Saavedra, L. A., Eggeling, C., & Barrantes, F. J. (2025). Concurrent diffusion of nicotinic acetylcholine receptors and fluorescent cholesterol disclosed by two-colour sub-millisecond MINFLUX-based single-molecule tracking. Nature Communications , 16(1), 1–16. https://doi.org/10.1038/S41467-025-61489-4;TECHMETA
In neurodegenerative disease research, lipid droplet metabolism is gaining recognition as a critical modulator of microglial immune function. These triglyceride-rich droplets are essential for regulating microglial activation, cytokine release, and phagocytosis. Their formation and turnover rate have also been identified as key factors for determining pro-inflammatory versus homeostatic microglial responses. In Alzheimer’s disease, intrinsic genetic risk factors such as the APOE4 genotype have been shown to induce undesirable accumulation of lipid droplets in microglia, raising concerns about promoting proper lipid droplet metabolism.
In a 2025 Cell Reportspublication, Stephenson et al. visualized and quantified changes in lipid metabolism in human induced pluripotent stem cell (iPSC)-derived microglia during immune activation. Biotium’s LipidSpot™ 488 Lipid Droplet Stain was used to detect changes in intracellular lipid droplets in response to lipopolysaccharide (LPS) and amyloid-beta exposure. This allowed the researchers to map lipid droplet accumulation and confirm its significant role in microglial immune signaling. Figure 1 below (courtesy of Stephenson, R.A.) shows an example of microglial staining using Biotium’s red fluorescent LipidSpot™ 610 from a related study.
Staining with LipidSpot™ 610 after the transduction of scrambled shRNA control or shRNA targeted knockdown of DGAT1 and DGAT2 (DGAT Knockdown) prevented lipid droplet biosynthesis in human induced pluripotent stem cell (iPSC)-derived microglia. Transduced microglia are indicated by GFP-positive cells. Microglia were treated with lipopolysaccharide (LPS) or vehicle for 16 hours. DGAT knockdown reduces the number of lipid droplets in DGAT KD GFP-positive microglia. Scale bars = 25 μm. Credit: Stephenson, R.A.
The authors also discovered that inhibition of triglyceride biosynthesis prevented LPS-induced microglial phagocytosis. Biotium’s ViaFluor® 488 SE was used as a reference cytoplasmic stain in the phagocytosis monitoring assay.
The study showed that pharmacological inhibition of triglyceride synthesis restored homeostatic gene expression, reduced NF-κB translocation, and improved surveillance deficits in APOE4-expressing microglia. Biotium’s lipid-specific and cytoplasmic fluorescent tools were instrumental in visualizing dynamic cellular changes. These discoveries highlight the potential of targeting lipid droplet metabolism to rebalance microglial immune function in Alzheimer’s disease.
Stephenson, R. A., Sepulveda, J., Johnson, K. R., Lita, A., Gopalakrishnan, J., Acri, D. J., Beilina, A., Cheng, L., Yang, L. G., Root, J. T., Ward, M. E., Combs, C., Skarnes, W. C., Cookson, M. R., Shih, H. Y., Larion, M., Rebeck, G. W., & Narayan, P. S. (2025). Triglyceride metabolism controls inflammation and microglial phenotypes associated with APOE4. Cell Reports, 44(7), 115961. https://doi.org/10.1016/j.celrep.2025.115961
Exosome-based therapies are gaining attention in regenerative medicine for their ability to modulate cellular behavior and enhance tissue repair. Exosomes derived from mesenchymal stem cells (MSCs) have been shown to carry bioactive molecules such as proteins, lipids, and nucleic acids that facilitate intercellular communication and tissue regeneration. While MSC-derived exosomes have demonstrated therapeutic potential for wound healing, clinical and veterinary exosome therapies remain limited due to challenges in producing sufficient quantities of high-quality exosomes in a scalable and reproducible manner.
In a 2025 Scientific Reportspublication, Pamulang et al. addressed these limitations and developed a novel upscaling protocol to isolate exosomes from canine adipose-derived MSCs (cAD-MSCs). This approach aimed to overcome the low exosome yield typical of conventional 2D culture systems while avoiding serum-derived contaminants by combining a microcarrier-based 3D culture system, a custom serum-free medium (VSCBIC-3), and tangential flow filtration. The protocol was evaluated not only for its ability to increase exosome production but also for preserving cell viability and producing vesicles with enhanced bioactivity.
To validate exosome purity and functionality, the team followed MISEV2023 guidelines for comprehensive characterization. Biotium’s AccuOrange™ Protein Quantification Kit was used to measure exosomal protein concentration for determining exosome yield. Expression of the exosomal surface marker CD9 was assessed using Biotium’s ExoBrite™ CD9 Flow Antibody on a CytoFLEX® LX Nanoscale Flow Cytometer. The study validated the scalability and therapeutic potential of cAD-MSC-derived exosomes for wound healing, advancing the potential for exosome therapies in veterinary clinical applications and future translational research.
Pamulang, Y. V., Oontawee, S., Rodprasert, W., Padeta, I., Sa-Ard-lam, N., Mahanonda, R., Osathanon, T., Somparn, P., Pisitkun, T., Torsahakul, C., & Sawangmake, C. (2025). Potential upscaling protocol establishment and wound healing bioactivity screening of exosomes isolated from canine adipose-derived mesenchymal stem cells. Scientific Reports 2025 15:1, 15(1), 1–20. https://doi.org/10.1038/s41598-025-93219-7
CytoFLEX® is a registered trademark of Beckman Coulter, Inc.
Fluorescence lifetime imaging microscopy (FLIM) has emerged as a powerful technique for multi-color imaging, allowing researchers to distinguish fluorochromes beyond traditional spectral separation methods. By leveraging fluorescence lifetime differences, FLIM provides additional fluorochrome discrimination, enabling more complex imaging experiments with fewer spectral constraints. This approach is particularly valuable in confocal and Stimulated Emission Depletion (STED) microscopy, where spectral overlap and bleed-through can limit the number of detectable fluorophores. Phasor analysis of fluorescence lifetimes offers a rapid and photon-efficient alternative to traditional curve-fitting methods, making it well-suited for imaging experiments with low signal-to-noise ratios or fast acquisition times.
In a 2025 Methods in Microscopy publication, Pisfil et al. used Biotium’s CF® Dyes CF®594 and CF®680R to validate fluorescence lifetime separation in both confocal and STED multi-color imaging. CF®680R was effectively distinguished from far-red ATTO647N and AS635P due to its shorter fluorescence lifetime, enabling clear separation in the phasor plot. WGA-CF®594 was used to stain cell surfaces and was successfully distinguished from Alexa Fluor Plus 594 in the same red channel (Figure 1 ). Their study demonstrated that FLIM could successfully separate two fluorochromes within a single spectral channel, increasing the number of distinguishable targets while maintaining imaging quality comparable to spectral separation methods.
Overlaid summary of 5-color confocal and STED images generated from 3 fluorescent channels with lifetime information. WGA CF®594 (orange) as separated from Alexa Fluor Plus 594 (white) in the first image under 5 color confocal; and CF®680R (blue) was used in the third confocal channel to detect nuclear pores and was cleared up from spectral bleed-through and noise. Credit: Pisfil et al., Methods in Microscopy (2025). Reproduced under CC BY 4.0.
Fluorescence lifetime separation is a promising advancement for multi-color fluorescence microscopy and can be optimized with the incorporation of Biotium’s bright and photostable CF® Dyes. As new fluorophores with broader lifetime ranges become available, FLIM may further improve the resolution and accuracy of cellular imaging studies. This technique offers great potential for investigating intricate biological processes, from cellular signaling to disease pathogenesis, in both fixed and live-cell imaging applications.
Pisfil, M. G., & Dietzel, S. (2025). Fluorochrome separation by fluorescence lifetime phasor analysis in confocal and STED microscopy. Methods in Microscopy, 2(1), 45–60. https://doi.org/10.1515/MIM-2024-0022
SpectraPlex by Leica Microsystems is a cutting-edge software integrated with the STELLARIS confocal platform that streamlines and enhances the workflow of 3D high-multiplex imaging. As highlighted in a Nature Portfolio Application Note, SpectraPlex enhances imaging accuracy with real-time unmixing algorithms that minimize artifacts, optimize signal detection, and eliminate iterative imaging cycles. This enables researchers to visualize multiple biological markers in a single session while maintaining sample integrity. Additionally, its innovative Virtual Fridge system streamlines high-multiplex experiments by organizing, storing, and managing fluorophore selections for reproducible panel development.
In another 2024 Nature Portfolio Application Note, Kunz et. al. of Leica Microsystems used SpectraPlex to build a 3D high-multiplex panel of a mouse pancreatic tumor section. The researchers used Biotium’s CF® Dyes to prepare primary antibody conjugates alpha-SMA-CF®568, FoxP3-CF®633, E-cadherin-CF®660C, and CD31-CF®800 as part of a one-shot, 15-plex workflow. This method captured a large set of targets while preserving both tissue structure and molecular integrity, unlike traditional sequential staining that can compromise samples and limit real-time adaptability of imaging strategies.
SpectraPlex also resolved overlapping fluorescence signals using a “Group” approach, staining three tissue sections with subsets of five markers each to create an unmixing matrix for the full 15-plex dataset. This minimized sample waste, enhanced signal separation, and enabled high-fidelity imaging of intricate tumor structures. By eliminating iterative imaging rounds, SpectraPlex avoided sample degradation and allowed the researchers to revisit regions of interest (ROIs) without introducing artifacts.
Biotium’s CF® Dyes complement SpectraPlex by delivering bright, photostable fluorescence with exceptional signal-to-noise, particularly in the near-IR range. Though four out of the 15 dyes used in the application note were CF® Dyes, as demonstrated in Figure 1b of this image from Nature Magazine, CF® Dyes are available in spectrally unique alternatives for all 15 colors. The synergy between SpectraPlex and CF® Dyes enhances spectral separation and ensures reliable, high-quality imaging for advancing cancer immunology research and diagnostics.
Learn more about Biotium’s bright and photostable CF® Dyes, now available in over 40 colors from blue to near-infrared.
Searching for the best CF® Dye that fits with your instrument configuration? Check out our handy CF® Dye Selection Tool to find the optimal CF® Dye for a specific laser/filter combination for microscopy, flow cytometry, or western blotting.
Full Citation:
Application Note: SpectraPlex: A powerful toolbox for advanced 3D high-multiplex imaging. (n.d.). https://www.nature.com/articles/d42473-024-00262-5
Application Note: 3D high-multiplex imaging in cancer immunology. (n.d.). https://www.nature.com/articles/d42473-024-00260-7
Mitochondria are essential for maintaining cellular health, with a key aspect of mitochondrial function being the mitochondrial membrane potential (mitochondrial potential). Mitochondrial potential is a valuable biomarker for detecting any disruptions linked to mitochondrial dysfunction. This is because it reflects the electrical gradient central to processes like ATP synthesis and calcium homeostasis across the inner mitochondrial membrane. These aspects make mitochondrial potential a useful metric for following apoptosis.
Rhodamine-based fluorescent probes such as TMRM, TMRE, and JC-1 are commonly used to measure mitochondrial membrane potential and work by accumulating within mitochondria in a mitochondrial potential-dependent manner. However, they have limited spectral compatibility and can reflect interference from factors other than mitochondrial potential (e.g., oxidative stress with JC-1). MitoView™ 633, part of Biotium’s MitoView™ catalog, is a far-red fluorescent dye specifically designed for mitochondrial imaging that has now been shown to have great potential in multiplexing and ΔΨm measurement, offering a promising imaging alternative to TMRM.
In a 2023 publication in Frontiers Physiology, Ernst et al. systematically characterized MitoView™ 633 and its performance in comparison to TMRM. Its spectral properties, mitochondrial staining specificity and photostability were evaluated, as well as its performance during mitochondrial potential depolarization and compatibility with other fluorescent markers. MitoView™ 633’s far-red emission reduces light scattering and enhances tissue penetration, making it advantageous for imaging in deeper tissue layers, and spectral analysis showed that it had superior thermal stability compared to TMRM. Confocal microscopy confirmed that MitoView™ 633 localized specifically within the mitochondrial matrix and exhibited minimal photobleaching, making it well-suited for extended imaging sessions. The dye also showed compatibility with co-staining alongside MitoSOX™ Red and Fluo-4 AM, enabling simultaneous imaging of complex mitochondrial and cellular processes in intact cells.
MitoView™ 633 was found to be a robust, versatile, and reliable tool for live-cell imaging of mitochondrial membrane potential, with unique spectral properties that enhance its compatibility with other dyes and its suitability for imaging in deeper tissues. Additionally, MitoView™ 633 proved advantageous for simplifying experimental workflows and reducing preparation time without requiring a washing step after dye loading. These features position MitoView™ 633 as a robust, versatile tool for studying mitochondrial dynamics and mitochondrial potential in live cells, with potential applications in both physiological and pathological contexts.
Characterization of MitoView™ 633 staining and fluorescence stability. (A, B): Representative confocal imaging showing colocalization of MitoView™ 633 (Ex/Em: 635 nm/664 nm) with MitoTracker Green (Ex/Em: 488 nm/516 nm) in adult cardiomyocytes (A) and iPSC-CMs (B). The line scanning imaging revealed overlapping between the peaks of these two fluorophores, confirming MitoView™ 633’s mitochondrial-specific staining. (C) MitoView™ 633 photobleaching measured at different concentrations. (D) Comparison of MitoView™ 633 photobleaching in adult cardiomyocytes and H9C2 cells. (E, F): Comparison of the thermo (E) and pH (F) fluorescence stability between MitoView™ 633 and TMRM. Each group includes 4-6 cells from 4 mice/cell cultures. (G, H): Effect of MitoView™ 633 loading concentration on cellular ATP content in adult cardiomyocytes (G) and AC16 cells (H). n = 4 per group. Credit: Ernst et al., Front. Physiol., 22 (2023).Reproduced under CC BY 4.0.
Learn more about Biotium’s wide range of mitochondrial dyes, in a wide variety of colors. Biotium also offers MitoView™ Fix 640, a far-red mitochondrial stain suitable for no-wash, long term staining in live cells that is well-retained after fixation, permeabilization, and subsequent immunofluorescence staining. Biotium’s Mitochondrial Marker Antibodies stain mitochondria in fixed cells or tissue sections and are available with a wide selection of bright and photostable CF® Dyes and other conjugations.
Full Citation
Ernst P, Kim S, Yang Z, Liu XM and Zhou L (2023) Characterization of the far-red fluorescent probe MitoView™ 633 for dynamic mitochondrial membrane potential measurement. Front. Physiol. 14:1257739. doi: 10.3389/fphys.2023.1257739
Triple-negative breast cancer (TNBC) is an aggressive cancer type with no specific therapeutic targets, leaving chemotherapy as the primary treatment option. However, chemotherapy is often highly toxic for patients with limited effectiveness, underscoring the need for new therapeutic strategies. Researchers are examining the potential of extracellular vesicles (EVs)- small, cell-derived particles that are naturally highly effective in intercellular communication- as a versatile platform for therapies beyond conventional drug delivery. EVs can be modified genetically or chemically to deliver therapeutic chemicals, peptides, or RNAs for higher cellular uptake in a targeted manner, even through challenging biological barriers like the blood-brain barrier. This selective targeting ability, combined with their endogenous origin, avoids triggering unwanted immune reactions and positions EVs as promising delivery vehicles for drugs.
In a 2024 publication in Molecular Therapy, Bhullar et al. engineered EVs using RNA nanotechnology to target TNBC cells specifically and inhibit tumor growth. EVs were loaded with a combination of chemotherapeutic drugs (gemcitabine or paclitaxel) and small interfering RNA (siRNA) designed to silence Survivin (SUR), an apoptosis inhibitor and known cancer target. Additionally, the EVs were decorated with a CD44 aptamer ligand to enhance their direct therapeutic targeting ability of TNBC cells and reduce off-target effects. The modified EVs were fluorescently labeled with Biotium’s ExoBrite™ CTB EV Staining Kits and were visualized with the fluorescent labeled RNA aptamer and chemically modified siRNA using super-resolved structured illumination microscopy (SR-SIM). These observations ensured that loading did not affect EV morphology and confirmed their structural integrity for precise encapsulation and targeted delivery.
The engineered EVs demonstrated promising results by effectively inhibiting tumor growth in a TNBC mouse model with significantly lower doses of gemcitabine and paclitaxel than standard chemotherapy. The successful therapeutic effects at lower doses highlight the potential of EV-based delivery systems to precisely target TNBC while reducing chemotherapy-associated toxicity and other adverse side effects. This study demonstrates an innovative interdisciplinary approach to cancer therapy that could improve patient outcomes through targeted, low-toxicity treatments.
Super-resolution dSTORM images of human colorectal cancer cell line-derived EVs. EVs were stained with anti-tetraspanin antibodies (an anti-CD9/CD63/CD81 cocktail, all conjugated to CF®647, shown in magenta) together with 1X ExoBrite™ 560/585 CTB (in cyan). Samples were prepared using ONI’s EV Profiler Kit and acquired using the Nanoimager S Mark II from ONI (Oxford Nanoimaging, UK). Data was processed using a beta-release version of CODI, ONI’s cloud-based data analysis platform. Scale bars are 500 nm (zoomed out, left panel) and 50 nm for single-EV panels (right). Image courtesy of ONI.
Bhullar AS, Jin K, Shi H, Jones A, Hironaka D, Xiong G, Xu R, Guo P, Binzel DW, Shu D. Engineered extracellular vesicles for combinatorial TNBC therapy: SR-SIM-guided design achieves substantial drug dosage reduction. Mol Ther. 2024 Oct 5:S1525-0016(24)00658-0. doi: 10.1016/j.ymthe.2024.09.034. Epub ahead of print. PMID: 39369270.
Stem-cell-based therapies, particularly ones utilizing human adipose-derived stem cells (ASCs), are becoming increasingly popular in the field of regenerative medicine. This is due to the relative ease of isolating ASCs from fat tissue compared to other stem cell subtypes. ASCs also play a role in modulating the immune system with anti-inflammatory cytokines and have the capacity to differentiate into cell types like fat, bone, and vascular tissue to promote regeneration in damaged, diseased, or aging cells. Despite the many promising characteristics of ASCs, their therapeutic potential is limited by reduced viability caused by their sensitivity to various stress factors that they are commonly exposed to during their use. These stressors include shear stress from needle injections, metabolic stress following transplantation, and exposure to DMSO during cryopreservation, which triggers increased reactive oxygen species (ROS) production leading to cell death.
Transgenic expression of proteins from extremophiles—organisms that thrive in extreme environments—are garnering attention for their potential to improve the stress tolerance of mammalian cells. Tardigrades are microscopic extremophiles that can enter a quiescent state capable of surviving radiation, desiccation, and extreme temperatures. They are a rich source of the mitochondrial abundant heat soluble (MAHS) protein, which has been shown to improve cell survival under osmotic stress without altering cell morphology.
In a 2024 publication in Scientific Reports, Rolsma et al. evaluated the transgenic expression of the MAHS protein in ASCs to determine whether it could enhance their tolerance to DMSO exposure, injection stress, and metabolic stress. MAHS-transgenic ASCs were stained with Biotium’s MitoView™ Fix 640 to confirm that the MAHS protein localized to mitochondria. The researchers assessed the effects of MAHS on the ASCs tolerance to freezing, DMSO, and passage through injection needles of varying diameters using Biotium’s Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells. The results suggest that ASCs expressing MAHS from tardigrades had significantly higher survival rates with needle shear stress and DMSO exposure. However, MAHS-transgenic ASCs showed no improvement in resistance to freezing, which contrasts with the natural freezing resistance observed in tardigrades. This highlights a limitation in the application of MAHS to cold storage or cryopreservation of ASCs. Lastly, the study assessed the effects of MAHS expression on ASC differentiation. The MAHS-transgenic ASCs preferentally differentiated toward osteogenic over adipogenic lineages.
The findings highlight the need for further research to fully understand the mechanisms by which MAHS influences cell differentiation and explore its application in other types of cells for regenerative medicine. By improving the resilience of stem cells to various stress factors, MAHS expression could pave the way for more reliable, efficient, and patient-friendly therapies.
Mitochondrial localization of MAHS. (A) Quantification of Pearson correlation coefficient for the overlap between expressed protein localization and mitochondrial morphology (two-sample t-test, n = 4). The denoted p-value indicates the pairwise difference in GFP/MitoView correlation between the genotypes. (B) Maximum projections of the overlap between expressed protein localization (AcGFP1 or AcGFP1-MAHS, cyan) and mitochondrial morphology (MitoView™, yellow) in ASC52telos transduced with either the AcGFP1 or AcGFP1-MAHS transgene. Scale bars are 25 µm. Credit: Rolsma et. al., Sci Rep 14, 11834 (2024). Reproduced under CC BY 4.0.
Rolsma, J.L., Darch, W., Higgins, N.C. et al. The tardigrade-derived mitochondrial abundant heat soluble protein improves adipose-derived stem cell survival against representative stressors. Sci Rep 14, 11834 (2024). https://doi.org/10.1038/s41598-024-62693-w
Cortical development in mammals is regulated by the coordination of cell proliferation, migration, and differentiation, processes reliant on microtubule organization. The protein ADP ribosylation factor-like GTPase 2 (Arl2) plays a crucial role in these mechanisms, particularly in its relationship with the centrosomal protein Cdk5rap2 towards microtubule growth and cortical development. While Arl2’s functions for microtubule growth have been described in fruit flies, its responsibilities and interactions in mammalian brain development have not been carefully studied.
In a 2024 publication in PLOS Biology, Ma et al. explored the role of Arl2 in regulating cortical development through the effects on neuronal migration and differentiation in Arl2 knockdown in mouse brains. Colocalization of CDK5rap2 and Arl2 was confirmed through co-immunoprecipitation and proximity ligation assays. Live-cell imaging using Biotium’s ViaFluor® Live Cell Microtubule Stains was also performed to assess the effects of Arl2 knockdown on cell cycle progression in mouse neural progenitor cells (mNPCs).
Arl2 knockdown was accomplished via transfection of shRNA targeting different regions of Arl2 in mNPCs and in vivo. Results show Arl2 knockdown caused neuronal migration defects and impaired cortical layering. Mitotic duration was also significantly extended in Arl2 knockdown mNPCs when compared to the control. Knockdown of Arl2 in vivo showed defects in neuronal proliferation and differentiation in the brain’s lower ventricular and subventricular zones (VZ and SVZ), and fewer neurons migrating towards the cortical plate (CP) to properly develop the cortex. Arl2 KD also reduced the length and number of neurites (the connective branches between neurons), further supporting that Arl2 is crucial for neuron maturation. In experiments where Arl2 was overexpressed (OE), an increase in neuronal migration was observed. Overexpression of a constitutively active Arl2Q70L mutant enhanced microtubule dynamics, while a dominant-negative Arl2T30N mutant led to impaired microtubule growth.
These findings establish Arl2 as critical for keeping neural progenitor cells alive to divide and migrate for proper brain cortex development. The importance of Arl2’s role emphasizes the need for closer analysis of its effects on neurodevelopmental disorders to develop potential therapeutic strategies.
Fig 4. Arl2 dysfunction results in defects in cell cycle progression in mNPCs. (A) Time series of mNPCs in vitro using Viafluor-647 live cell microtubule staining kit (Biotium, #70063) in shCtrl and shArl2. (B) Quantification graph showing the average time taken for a single mitotic cycle in control = 1.32 ± 0.21 h and shArl2 = 6.55 ± 1.52 h. Credit: Modified from Ma etl al., PLoS Biol 22(8): e3002751. reproduced under CC BY 4.0.
Full Citation: Ma D, Lin K-Y, Suresh D, Lin J, Gujar MR, Aung HY, et al. (2024) Arl2 GTPase associates with the centrosomal protein Cdk5rap2 to regulate cortical development via microtubule organization. PLoS Biol 22(8): e3002751. https://doi.org/10.1371/journal.pbio.3002751
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