Living Animal Cells Research Articles

Monitoring Circulating Myeloid Cells in Peritonitis with an In Vivo Imaging Flow Cytometer.

Peritonitis is a common and life-threatening inflammatory disease. Myeloid cells are elevated in the peripheral blood and contribute to peritonitis, but their circulating dynamics are not clear. In vivo flow cytometry (IVFC) is a noninvasive technique for monitoring the dynamics of circulating cells in live animals. It has been extensively used to detect circulating tumor cells, but rarely for monitoring immune cells. Here, we describe a method adapting an intravital microscope for IVFC so that we can monitor LysM-EGFP-labeled circulating myeloid cells in a tumor necrosis factor (TNF) α-induced peritonitis mouse model. Using this IVFC method, we quantified the blood flow velocity and cell concentration in circulation. We observed a significant increase in LysM-EGFP+ cells in circulation after TNFα intraperitoneal (i.p.) injection, which reached a plateau in ~20 min. Conventional cytometry analysis showed that most LysM-EGFP+ cells were neutrophils. Increasing blood neutrophils were accompanied by neutrophil recruitment to the peritoneal cavity and neutrophil emigration from the bone marrow. We then monitored neutrophil CD64 expression in vivo and found a significant increase in TNFα-induced peritonitis. We also found that CD18 blockade doubled the circulating neutrophil number in TNFα-induced peritonitis, suggesting that CD18 is critical for neutrophil recruitment in peritonitis. Overall, we demonstrate that IVFC techniques are useful for studying the circulating dynamics of immune cells during inflammatory diseases.

Abstract P1066: Engineering Of Ferritin Overexpressing Human Induced Pluripotent Stem Cells For In Vivo Tracking Of Grafts Using Magnetic Resonance Imaging

Introduction: Transplantation of human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) emerges as a promising approach for the treatment of heart diseases. However, it is an unmet challenge to precisely localize the hiPSC-CMs upon transplantation and quantify the graft size in the live large animals. To address this issue, we established a new approach to track grafted CMs in large animals using magnetic resonance imaging detectable reporter molecules, which allows the assessment of graft size in live animals at different time points. Methods: We employed a CRISPR/Cas9 system as a virus-free approach to overexpress ferritin in hiPSCs. Results: hiPSC cells were transfected with ferritin heavy chain CRISPR activation plasmid for 24 hours. Before antibiotic selection, we established a kill curve to determine the appropriate antibiotic concentration. Based on the kill curve, we conducted antibiotic selection using puromycin for 7 days and isolated transfected colonies for further culture. Next, we treated various concentrations of iron chloride (0-100 μM) to the FHC transfected hiPSC for 4 hours to examine a proper concentration of iron loading for MRI detection. There was no significant difference in cell viability between 0 and 25 μM of iron chloride. In addition, we confirmed transfection efficiency with higher ferritin expression in transfected cells compared to controls (non-transfected wild-type hiPSC). Following, we plan to differentiate ferritin overexpressing hiPSC into cardiomyocytes, inject the cells into porcine hearts, and conduct magnetic resonance imaging to detect the engrafted transgenic cells in live animals. Conclusion: The ferritin-expressing hiPSC line has the potential to provide a reliable measurement of the graft size in large animals.

(Invited, Digital Presentation) Donor-Acceptor-Linked Molecules for Controlling the Membrane Potential of Living Cells

Living animal cells have the plasma membrane potential (V m), which results from a difference in the electrical charge on the two extreme sides of the plasma membrane, that is, a slight excess of positive and negative ions outside and inside a cell, respectively. Such a difference is controlled by various ion channels. In neuronal cells, the V m is negative in the resting state and is drastically changed for neurotransmission. In response to some input, the ion channels of neuronal cells get quickly and transiently activated such that the abovementioned difference is cancelled (∆V m > 0, depolarization). Coordinated activation and deactivation of the ion channels occurs along the neuronal axons, which generates electrical pulses. This is the mechanism of neurotransmission. Therefore, the control of the V m using a light source is an attractive strategy that can allow targeted, fast control of neuronal activities. In this symposium, we have demonstrated a novel general strategy for the control of the V m by using donor-acceptor-linked molecules (D-A molecules) capable of intramolecular long-lived charge separation, nanocarriers for membrane targeting of D-A molecules, and light.The D-A molecules used in this study were triadic complexes of ferrocene (Fc), porphyrin (H2P), and fullerene (C60) (Fc-H2P-C60). We had previously reported that, under light irradiation, Fc-H2P-C60 yields the ferrocenium cation (Fc+)-H2P-C60 radical anion (C60 •-) with a long lifetime of ~0.01 ms [1]. Fc-H2P-C60 was solubilized in the physiological media with a genetically engineered lipoprotein nanoparticle (cpHDL). cpHDL was capable of high plasma membrane binding without significant cytotoxicity [2]. The Fc-H2P-C60/cpHDL complex was added to a cell culture medium of neuronal cells (rat pheochromocytoma PC12), and its plasma membrane binding was confirmed by confocal analysis and freeze-fracture electron microscopy. Subsequent light irradiation of PC12 cells decreased the membrane potential (∆V m > 0, depolarization). Such depolarization was not observed for the reference molecule (H2P alone) that was incapable of charge separation, clearly demonstrating that the charge separation was responsible for this depolarization [3]. Our following study with other D-A molecules revealed that the degree of the depolarization was positively correlated with the charge separation yield [4].To gain insight into the mechanism of this photo-induced depolarization, cytotoxicity, channel activities, and membrane capacitance were evaluated in D-A molecule-treated PC12 cells under or after irradiation. Pore formation in the plasma membrane, resulting from severe membrane damage, were not detected. Indeed, the photo-generation of reactive oxygen species (ROS) leading to membrane disruption was less efficient in the D-A molecules than in H2P incapable of photo-induced charge separation. In contrast, it was found that a specific subtype of cation channels was inhibited, and that the membrane capacitance was decreased by irradiated D-A molecules [5]. In this symposium, I will discuss more about the potential of applying our D-A molecules/carriers system in the biomedical fields.REFERENCES[1] Imahori, H. et al., J. Am. Chem. Soc. 2001, 123, 2607-2617.[2] Nakatsuji, H. et al., Angew. Chem. Int. Ed. 2015, 54, 11725-11729.[3] Numata, T. et al., J. Am. Chem. Soc. 2012, 134, 6092-6095.[4] Takano, Y. et al., Chem. Sci. 2016, 7, 3331-3337.[5] Numata, T. et al., Cell. Physiol. Biochem. 2020, 54, 899-916.

Magnetic Nano-Platform Enhanced iPSC-Derived Trabecular Meshwork Delivery and Tracking Efficiency.

PurposeTransplantation of stem cells to remodel the trabecular meshwork (TM) has become a new option for restoring aqueous humor dynamics and intraocular pressure homeostasis in glaucoma. In this study, we aimed to design a nanoparticle to label induced pluripotent stem cell (iPSC)-derived TM and improve the delivery accuracy and in vivo tracking efficiency.MethodsPLGA-SPIO-Cypate (PSC) NPs were designed with polylactic acid-glycolic acid (PLGA) polymers as the backbone, superparamagnetic iron oxide (SPIO) nanoparticles, and near-infrared (NIR) dye cypate. In vitro assessment of cytotoxicity, iron content after NPs labeling, and the dual-model monitor was performed on mouse iPSC-derived TM (miPSC-TM) cells, as well as immortalized and primary human TM cells. Cell function after labeling, the delivery accuracy, in vivo tracking efficiency, and its effect on lowering IOP were evaluated following miPSC-TM transplantation in mice.ResultsInitial in vitro experiments showed that a single-time nanoparticles incubation was sufficient to label iPSC-derived TM and was not related to any change in both cell viability and fate. Subsequent in vivo evaluation revealed that the use of this nanoparticle not only improves the delivery accuracy of the transplanted cells in live animals but also benefits the dual-model tracking in the long term. More importantly, the use of the magnet triggers a temporary enhancement in the effectiveness of cell-based therapy in alleviating the pathologies associated with glaucoma.ConclusionThis study provided a promising approach for enhancing both the delivery and in vivo tracking efficiency of the transplanted cells, which facilitates the clinical translation of stem cell-based therapy for glaucoma.

Non-Destructive Monitoring via Electrochemical NADH Detection in Murine Cells.

Nicotinamide adenine dinucleotide (NADH) is an important cofactor involved in metabolic redox reactions in living cells. The detection of NADH in living animal cells is a challenge. We developed a one-step monitoring method for NADH via an electrocatalytic reaction that uses a surface-modified, screen-printed electrode (SPE) having a redox active monolayer 4′-mercapto-N-phenlyquinone diamine (NPQD) formed by a self-assembled monolayer (SAM) of an aromatic thiol, 4-aminothiophenol (4-ATP). This electrode has a limit of detection (LOD) of 0.49 μM and a sensitivity of 0.0076 ± 0.0006 μM/μA in cell culture media, which indicates that it retains its selectivity. The applicability of this NADH sensor was demonstrated for the first time by cell viability monitoring via NADH-sensing in cell culture supernatants.

Orange/red dual-emissive boron- and nitrogen-codoped carbon dots for wash-free and selective staining of lipid droplets in live cells

Lipid droplet (LD) is an organelle that stores lipids and plays crucial roles in many metabolic processes and diseases. However, developing a method for long-term tracing of LDs and monitoring LD movement remains a challenge. Here, by using a solvent-free carbonization method, we prepare a kind of boron- and nitrogen-codoped carbon dots (BNCDs) from 3-aminophenylboronic acid. The as-synthesized BNCDs have an average size of ∼6.9 nm, long-wavelength fluorescence emission, and excitation wavelength-independent fluorescence emission, and can realize wash-free LD imaging without the introduction of LD-targeting ligands. Besides, the BNCDs have the characteristics of good biocompatibility, simple preparation, and low cost. More importantly, the BNCDs can effectively track the dynamic changes of LDs (including lipophagy) in live animal cells. This work shows that the developed BNCDs can be used as an ideal probe for nondestructively visualizing LDs, which may have implications for the future studies on LDs-related metabolism and diseases.

A biocompatible two-photon absorbing fluorescent mitochondrial probe for deepin vivobioimaging

Mitochondria, key organelles which keep in tune with energy demands for eukaryotic cells, are firmly associated with neurological conditions and post-traumatic rehabilitation. In vivo fluorescence imaging of mitochondria, especially with deep tissue penetration, would open a window to investigate the actual context of the brain. However, the depth of traditional two-photon mitochondrial fluorescence imaging is still limited due to the poor biological compatibility or low two-photon absorption cross-sections. A biocompatible mitochondria-targeted two-photon fluorescent dye (FO2) with an excellent two-photon absorption cross-section (the maximum of 1184 GM at 790 nm) and low cellular toxicity was designed and synthesized to overcome this problem. With this dye, we reached an imaging depth of ca. 640 μm during mitochondrial imaging of cortical cells in live animals. FO2 could be an excellent mitochondrial probe for live animal neural imaging to investigate the function and dysfunction of mitochondria in the brain.

Two-photon activated precision molecular photosensitizer targeting mitochondria

Mitochondria metabolism is an emergent target for the development of novel anticancer agents. It is amply recognized that strategies that allow for modulation of mitochondrial function in specific cell populations need to be developed for the therapeutic potential of mitochondria-targeting agents to become a reality in the clinic. In this work, we report dipolar and quadrupolar quinolizinium and benzimidazolium cations that show mitochondria targeting ability and localized light-induced mitochondria damage in live animal cells. Some of the dyes induce a very efficient disruption of mitochondrial potential and subsequent cell death under two-photon excitation in the Near-infrared (NIR) opening up possible applications of azonia/azolium aromatic heterocycles as precision photosensitizers. The dipolar compounds could be excited in the NIR due to a high two-photon brightness while exhibiting emission in the red part of the visible spectra (600–700 nm). Interaction with the mitochondria leads to an unexpected blue-shift of the emission of the far-red emitting compounds, which we assign to emission from the locally excited state. Interaction and possibly aggregation at the mitochondria prevents access to the intramolecular charge transfer state responsible for far-red emission.

Membrane dynamics of ATG4B and LC3 in autophagosome formation

The biogenesis of autophagosomes provides the basis for macroautophagy to capture and degrade intracellular cargoes. Binding of the autophagy-related protein ATG8/LC3 to autophagic membranes is essential to autophagosome formation, which involves the specific and dynamic processing of ATG8/LC3 by cysteine protease ATG4. However, to date, the mechanism whereby ATG4 is recruited to the membranes, the interaction of ATG4 and ATG8/LC3 on the membranes, and its role in the growth of phagophore are not completely understood. Here, we used fluorescence recovery after photobleaching to monitor the turnover of GFP-tagged ATG4B and LC3B in living animal cells. The data show that ATG4B localizes to early autophagic membranes in an LC3B-dependent manner. During autophagy, ATG4B and LC3B undergo rapid cytosol/isolation membrane exchange but not at the cytosol/completed autophagosome. In addition, ATG4B activity controls the efficiency of autophagosome formation by impacting the membrane binding/dissociation of LC3B. These data suggest that ATG4 and LC3 play interdependent roles in the formation of autophagosomes.

Quantitative imaging of RNA polymerase II activity in plants reveals the single-cell basis of tissue-wide transcriptional dynamics.

The responses of plants to their environment are often dependent on the spatiotemporal dynamics of transcriptional regulation. While live-imaging tools have been used extensively to quantitatively capture rapid transcriptional dynamics in living animal cells, the lack of implementation of these technologies in plants has limited concomitant quantitative studies in this kingdom. Here, we applied the PP7 and MS2 RNA-labelling technologies for the quantitative imaging of RNA polymerase II activity dynamics in single cells of living plants as they respond to experimental treatments. Using this technology, we counted nascent RNA transcripts in real time in Nicotiana benthamiana (tobacco) and Arabidopsis thaliana. Examination of heat shock reporters revealed that plant tissues respond to external signals by modulating the proportion of cells that switch from an undetectable basal state to a high-transcription state, instead of modulating the rate of transcription across all cells in a graded fashion. This switch-like behaviour, combined with cell-to-cell variability in transcription rate, results in mRNA production variability spanning three orders of magnitude. We determined that cellular heterogeneity stems mainly from stochasticity intrinsic to individual alleles instead of variability in cellular composition. Together, our results demonstrate that it is now possible to quantitatively study the dynamics of transcriptional programs in single cells of living plants.

Stochastic contraction of myosin minifilaments drives evolution of microridge protrusion patterns in epithelial cells.

Actin-based protrusions vary in morphology, stability, and arrangement on cell surfaces. Microridges are laterally elongated protrusions on mucosal epithelial cells, where they form evenly spaced, mazelike patterns that dynamically remodel by fission and fusion. To characterize how microridges form their highly ordered, subcellular patterns and investigate the mechanisms driving fission and fusion, we imaged microridges in the maturing skin of zebrafish larvae. After their initial development, microridge spacing and alignment became increasingly well ordered. Imaging F-actin and non-muscle myosin II (NMII) revealed that microridge fission and fusion were associated with local NMII activity in the apical cortex. Inhibiting NMII blocked fission and fusion rearrangements, reduced microridge density, and altered microridge spacing. High-resolution imaging allowed us to image individual NMII minifilaments in the apical cortex of cells in live animals, revealing that minifilaments are tethered to protrusions and often connect adjacent microridges. NMII minifilaments connecting the ends of two microridges fused them together, whereas minifilaments oriented perpendicular to microridges severed them or pulled them closer together. These findings demonstrate that as cells mature, cortical NMII activity orchestrates a remodeling process that creates an increasingly orderly microridge arrangement.

(Invited) Optical Control of the Membrane Potential of Living Cells with Donor-Acceptor-Linked Molecules

In living animal cells, there exists a difference in the electrical charge on the two extreme sides of the plasma membrane, that is, a slight excess of positive and negative ions outside and inside a cell, respectively. Such a charge difference produces a membrane potential (V m) across the plasma membrane. The V m (< 0) is controlled by various ion channels, and, especially, in neuronal cells, it is drastically changed for neurotransmission. In response to some input, the ion channels of neuronal cells get quickly activated such that the abovementioned difference is cancelled (∆V m > 0, depolarization). This electrical signal propagates along the neuronal axons like a wave through coordinated activation and deactivation of the ion channels. Therefore, the control of membrane potential and ion transport across the plasma membrane using a light source is an attractive strategy that can allow targeted, fast control of precisely defined events in the plasma membrane. In this symposium, we have demonstrated a novel general strategy for the control of membrane potential and ion transport by using donor-acceptor-linked molecules (D-A molecules) capable of intramolecular long-lived charge separation, nanocarriers for solubilization, membrane targeting of D-A molecules, and light (Figure 1).For the preparation of the D-A molecule, porphyrin (H2P) derivatives with ferrocene (Fc) and fullerene (C60) were synthesized to create a triadic arrangement (Fc-H2P-C60). We had previously reported that, under light irradiation, Fc-H2P-C60 yields the ferrocenium cation (Fc+)-H2P-C60 radical anion (C60 •-) with a long lifetime of ~0.01 ms [1]. For its solubilization in the physiological media and safer plasma membrane delivery, we focused on a lipoprotein in our body considering its amphiphilic property. High-density lipoprotein (HDL) can be reconstituted with recombinant lipid-binding proteins and phospholipids. We had previously reported that HDL genetically and chemically engineered with cytophilic peptides and fusogenic lipids is capable of high plasma membrane binding without significant cytotoxicity [2]. For this purpose, Fc-H2P-C60 was solubilized with one of our engineered HDL nanoparticles (cpHDL) and added to a cell culture medium of rat pheochromocytoma cells (PC12). The cell membrane delivery of Fc-H2P-C60 was suggested by confocal analysis and freeze-fracture electron microscopy. Subsequent light irradiation of PC12 cells increased the membrane potential (depolarization), which was not observed for the reference molecule (H2P) that was incapable of charge separation [3]. In addition, the inhibition of potassium ion flow across the membrane was found to be responsible for this depolarization (Figure 1). Our following study with 3 D-A molecules, including Fc-H2P-C60, revealed that the degree of the depolarization was positively correlated with the charge separation yield [4]. In this symposium, I will discuss more about the potential of applying our D-A molecules/carriers system in the biomedical fields.Figure 1. Schematic illustration of optical control of membrane potential and cation flow using a D-A molecule (Fc-H2P-C60)[1] Imahori, H. et al., J. Am. Chem. Soc. 2001, 123, 2607-2617.[2] Murakami, T., Biotechncol. J. 2012, 7, 762-767.; Kim, H. et al. Biochim. Biophys. Acta Biomembr. 2019, 1861, 183008.[3] Numata, T. et al., J. Am. Chem. Soc. 2012, 134, 6092-6095.[4] Takano, Y. et al., Chem. Sci. 2016, 7, 3331-3337. Figure 1

Tracking the fate and migration of cells in live animals with cell-cycle indicators and photoconvertible proteins

Cell migration and cell proliferation are the basic principles that make up a living organism, and both biologically and medically. In order to understand living organism and biological phenomena, it is essential to track the migration, proliferation, and fate of cells in living cells and animals and to clarify the properties and molecular expression of cells. Recent developments in novel fluorescent proteins have made it possible to observe cell migration and proliferation as the cell cycle at the single-cell level in living individuals and tissues. Here, we introduce cell cycle visualization of living cells and animals by Fucci (Fluorescent Ubiquitination-based Cell Cycle Indicator) system and in situ cell labeling of cells and tracking cell migration by photoactivatable and photoconvertible proteins. In addition, we will present our established methods as an example of combines above tools with single-cell molecular expression analysis to reveal the fate of migrating cells at single cell level.

A practical guide to photoswitching optoacoustics tomography.

Photochromic proteins and photoswitching optoacoustics (OA) are a promising combination, that allows OA imaging of even small numbers of cells in whole live animals and thus can facilitate a more wide-spread use of OA in life-science and preclinical research. The concept relies on exploiting the modulation achieved by the photoswitching to discriminate the agents' signal from the non-modulating background. Here we share our analysis approaches that can be readily used on data generated with commercial OA tomography imaging instrumentation allowing-depending on the used photoswitching agent and sample-routine visualizations of as little as several hundreds of transgene labeled cells per imaging volume in the live animal.

Should I Stay or Should I Flow: HSCs Are on the Move!

Stem cell biologists have been yearning to visualize hematopoietic stem cells (HSCs) in live animals since Kiel etal. (2005) first visualized them in bone cavities. With two recent papers from Christodoulou etal. (2020) and Upadhaya etal. (2020), we can all now see how HSCs behave in their niches!

Recent advances in intravital microscopy for investigation of dynamic cellular behavior in vivo

Currently, most biological research relies on conventional experimental techniques that allow only static analyses at certain time points in vitro or ex vivo. However, if one could visualize cellular dynamics in living organisms, that would provide a unique opportunity to study key biological phenomena in vivo. Intravital microscopy (IVM) encompasses diverse optical systems for direct viewing of objects, including biological structures and individual cells in live animals. With the current development of devices and techniques, IVM addresses important questions in various fields of biological and biomedical sciences. In this mini-review, we provide a general introduction to IVM and examples of recent applications in the field of immunology, oncology, and vascular biology. We also introduce an advanced type of IVM, dubbed real-time IVM, equipped with video-rate resonant scanning. Since the real-time IVM can render cellular dynamics with high temporal resolution in vivo, it allows visualization and analysis of rapid biological processes.

Novel hybrids based on graphene quantum dots covalently linked to glycol corroles for multiphoton bioimaging

Graphene quantum dots (GQDs) possess excellent optical properties, high photostability, aqueous solubility and bio-compatibility. The presence of carboxyl and hydroxyl groups on GQDs surface and edges provides an excellent opportunity to explore them as anchoring units for covalent functionalization. In addition, the nonlinear optical response of GQDs allows the development of optical sensors operating in biological media. To evaluate the possibility of using GQDs to introduce a hydrophobic sensing unit inside animal cells and to add a nonlinear response to the sensing unit we prepared a novel hybrid based on GQDs covalently linked to corrole units bearing a glycol branch. Covalent functionalization was supported by X-ray photoelectron spectroscopy (XPS), Raman and Fourier transform infrared (FTIR) spectroscopy. Both the height and the diameter of the hybrid showed distributions peaking below 4 nm. The UV–vis absorption and emission spectra of the hybrids were additive with respect to that of the GQDs and the corrole. Insights about the most stable conformation of the corrole with respect to the GQDs core was provided by in silico studies using a model structure. The internalization and distribution of the hybrids in live animal cells was evaluated in human breast adenbocarcinoma cell line (MCF-7 cells) using confocal and multiphoton microscopy. Notably, multiphoton microscopy allowed for image collection using nonlinear excitation in the near-infrared and emission in the red part of the visible spectra.

Effect of Sulphonylurea Derivatives and Short Chain Fatty Acids on Expression of Incretins Hormone in living Animal Cells

Dietary fibres are a type of carbohydrates that are found in plant-based foods. They are not absorbed or digested by the body but play an important role in maintaining good health. Short-chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate, are metabolites formed by gut microbiota from these complex dietary carbohydrates. The formed acids have a role in the secretion of hormones such as glucagon-like peptide-1(GLP-1) and glucose-dependent insulin tropic polypeptide (GIP) that help the body to control diabetes. Diabetes mellitus is a chronic disease that occurs when the pancreas fails to produce enough insulin, or when the body cannot use it effectively. The hormones GLP-1 and GIP, which secreted after a meal, like other enteroendocrine hormones help to orchestrate the body's response to the availability of newly absorbable nutrients and are noteworthy, stimulate postprandial insulin secretion. This study clarifies the effects and the roles of organic compounds such as Short Chain Fatty Acids (SCFAs) and sulphonylurea derivatives on body glucose homeostasis and clarifies the effects of both SCFAs and sulphonylurea on incretin expression by investigating the expression of GLP-1 and GIP hormones in tissues and blood samples.

Cybrid technology.

The study of the mitochondrial DNA (mtDNA) has been hampered by the lack of methods to genetically manipulate the mitochondrial genome in living animal cells. This limitation has been partially alleviated by the ability to transfer mitochondria (and their mtDNAs) from one cell into another, as long as they are from the same species. This is done by isolating mtDNA-containing cytoplasts and fusing these to cells lacking mtDNA. This transmitochondrial cytoplasmic hybrid (cybrid) technology has helped the field understand the mechanism of several pathogenic mutations. In this chapter, we describe procedures to obtain transmitochondrial cybrids.

Direct observation of branching MT nucleation in living animal cells.

Centrosome-mediated microtubule (MT) nucleation has been well characterized; however, numerous noncentrosomal MT nucleation mechanisms exist. The branching MT nucleation pathway envisages that the γ-tubulin ring complex (γ-TuRC) is recruited to MTs by the augmin complex to initiate nucleation of new MTs. While the pathway is well conserved at a molecular and functional level, branching MT nucleation by core constituents has never been directly observed in animal cells. Here, multicolor TIRF microscopy was applied to visualize and quantitatively define the entire process of branching MT nucleation in dividing Drosophila cells during anaphase. The steps of a stereotypical branching nucleation event entailed augmin binding to a mother MT and recruitment of γ-TuRC after 15 s, followed by nucleation 16 s later of a daughter MT at a 36° branch angle. Daughters typically remained attached throughout their ∼40-s lifetime unless the mother depolymerized past the branch point. Assembly of branched MT arrays, which did not require Drosophila TPX2, enhanced localized RhoA activation during cytokinesis.