Generating stable isolated mitochondrial recipient clones in mammalian cells using MitoPunch mitochondrial transfer.

This protocol describes the assembly and use of MitoPunch to deliver mitochondria containing mitochondrial DNA (mtDNA) into cells lacking mtDNA (ρ0 cells). MitoPunch generates stable isolated mitochondrial recipient clones with restored mtDNA and recovered respiration, enabling investigation of mtDNA mutations and mtDNA-nuclear DNA interactions in a range of cell types. For complete details on the use and execution of this protocol, please refer to Sercel et al. (2021) and Patananan et al. (2020).

Stable transplantation of human mitochondrial DNA by high-throughput, pressurized isolated mitochondrial delivery.

Generating mammalian cells with specific mitochondrial DNA (mtDNA)-nuclear DNA (nDNA) combinations is desirable but difficult to achieve and would be enabling for studies of mitochondrial-nuclear communication and coordination in controlling cell fates and functions. We developed 'MitoPunch', a pressure-driven mitochondrial transfer device, to deliver isolated mitochondria into numerous target mammalian cells simultaneously. MitoPunch and MitoCeption, a previously described force-based mitochondrial transfer approach, both yield stable isolated mitochondrial recipient (SIMR) cells that permanently retain exogenous mtDNA, whereas coincubation of mitochondria with cells does not yield SIMR cells. Although a typical MitoPunch or MitoCeption delivery results in dozens of immortalized SIMR clones with restored oxidative phosphorylation, only MitoPunch can produce replication-limited, non-immortal human SIMR clones. The MitoPunch device is versatile, inexpensive to assemble, and easy to use for engineering mtDNA-nDNA combinations to enable fundamental studies and potential translational applications.

Mitochondria are specialized structures within cells that generate vital energy and biological building blocks. Mitochondria have a double membrane and contain many copies of their own circular DNA (mitochondrial DNA), which include the blueprints to create just thirteen essential mitochondrial proteins. Like all genetic material, mitochondrial DNA can become damaged or mutated, and these changes can be passed on to offspring. Some of these alterations are linked to severe and debilitating diseases. Both the double membrane of the mitochondria and their high number of DNA copies make treating such diseases difficult. A successful therapy must be capable of correcting almost every copy of mitochondrial DNA. However, the multiple copies of mitochondrial DNA create a problem for genetic research as current techniques are unable to reliably introduce particular mitochondrial mutations to all types of human cells to investigate how they may alter cell function. Sercel, Patananan et al. have developed a method to deliver new mitochondria into thousands of cells at the same time. This technique, called MitoPunch, uses a pressure-driven device to propel mitochondria taken from donor cells into recipient cells without mitochondrial DNA to reestablish their function. Using human cancer cells and healthy skin cells that lack mitochondrial DNA, Sercel, Patananan et al. showed that cells that received mitochondria retained the new mitochondrial DNA. The technique uses readily accessible parts, meaning it can be performed quickly and inexpensively in any laboratory. It further only requires a small amount of donor starting material, meaning that even precious samples with limited material could be used as mitochondrial donors. This new technique has several important potential applications for mitochondrial DNA research. It could be used in the lab to create large numbers of cell lineswith known mutations in the mitochondrial DNA to establish new systems that test drugs or probe the interaction between mitochondrial and nuclear DNA. It could be used to study a broad spectrum of biological questions since mitochondrial function is essential for several processes required for life. Critically, it could also be used as a starting point to develop next-generation therapies capable of treating inherited mitochondrial genetic diseases in severely affected patients.

Pressure-Driven Mitochondrial Transfer Pipeline Generates Mammalian Cells of Desired Genetic Combinations and Fates.

Generating mammalian cells with desired mitochondrial DNA (mtDNA) sequences is enabling for studies of mitochondria, disease modeling, and potential regenerative therapies. MitoPunch, a high-throughput mitochondrial transfer device, produces cells with specific mtDNA-nuclear DNA (nDNA) combinations by transferring isolated mitochondria from mouse or human cells into primary or immortal mtDNA-deficient (ρ0) cells. Stable isolated mitochondrial recipient (SIMR) cells isolated in restrictive media permanently retain donor mtDNA and reacquire respiration. However, SIMR fibroblasts maintain a ρ0-like cell metabolome and transcriptome despite growth in restrictive media. We reprogrammed non-immortal SIMR fibroblasts into induced pluripotent stem cells (iPSCs) with subsequent differentiation into diverse functional cell types, including mesenchymal stem cells (MSCs), adipocytes, osteoblasts, and chondrocytes. Remarkably, after reprogramming and differentiation, SIMR fibroblasts molecularly and phenotypically resemble unmanipulated control fibroblasts carried through the same protocol. Thus, our MitoPunch "pipeline" enables the production of SIMR cells with unique mtDNA-nDNA combinations for additional studies and applications in multiple cell types.

Modifying the Mitochondrial Genome.

Human mitochondria produce ATP and metabolites to support development and maintain cellular homeostasis. Mitochondria harbor multiple copies of a maternally inherited, non-nuclear genome (mtDNA) that encodes for 13 subunit proteins of the respiratory chain. Mutations in mtDNA occur mainly in the 24 non-coding genes, with specific mutations implicated in early death, neuromuscular and neurodegenerative diseases, cancer, and diabetes. A significant barrier to new insights in mitochondrial biology and clinical applications for mtDNA disorders is our general inability to manipulate the mtDNA sequence. Microinjection, cytoplasmic fusion, nucleic acid import strategies, targeted endonucleases, and newer approaches, which include the transfer of genomic DNA, somatic cell reprogramming, and a photothermal nanoblade, attempt to change the mtDNA sequence in target cells with varying efficiencies and limitations. Here, we discuss the current state of manipulating mammalian mtDNA and provide an outlook for mitochondrial reverse genetics, which could further enable mitochondrial research and therapies for mtDNA diseases.

Mitochondrial Transfer by Photothermal Nanoblade Restores Metabolite Profile in Mammalian Cells.

mtDNA sequence alterations are challenging to generate but desirable for basic studies and potential correction of mtDNA diseases. Here, we report a new method for transferring isolated mitochondria into somatic mammalian cells using a photothermal nanoblade, which bypasses endocytosis and cell fusion. The nanoblade rescued the pyrimidine auxotroph phenotype and respiration of ρ0 cells that lack mtDNA. Three stable isogenic nanoblade-rescued clones grown in uridine-free medium showed distinct bioenergetics profiles. Rescue lines 1 and 3 reestablished nucleus-encoded anapleurotic and catapleurotic enzyme gene expression patterns and had metabolite profiles similar to the parent cells from which the ρ0 recipient cells were derived. By contrast, rescue line 2 retained a ρ0 cell metabolic phenotype despite growth in uridine-free selection. The known influence of metabolite levels on cellular processes, including epigenome modifications and gene expression, suggests metabolite profiling can help assess the quality and function of mtDNA-modified cells.

Direct Nuclear Delivery of DNA by Photothermal Nanoblade.

We demonstrate direct nuclear delivery of DNA into live mammalian cells using the photothermal nanoblade. Pulsed laser-triggered cavitation bubbles on a titanium-coated micropipette tip punctured both cellular plasma and nuclear membranes, which was followed by pressure-controlled delivery of DNA into the nucleus. High-level and efficient plasmid expression in different cell types with maintained cell viability was achieved.

Massively parallel delivery of large cargo into mammalian cells with light pulses.

We report a high-throughput platform for delivering large cargo elements into 100,000 cells in 1 min. Our biophotonic laser-assisted surgery tool (BLAST) generates an array of microcavitation bubbles that explode in response to laser pulsing, forming pores in adjacent cell membranes through which cargo is gently driven by pressurized flow. The platform delivers large items including bacteria, enzymes, antibodies and nanoparticles into diverse cell types with high efficiency and cell viability. We used this platform to explore the intracellular lifestyle of Francisella novicida and discovered that the iglC gene is unexpectedly required for intracellular replication even after phagosome escape into the cell cytosol.

Effects of extracellular polymeric substances on the bioaccumulation of mercury and its toxicity toward the cyanobacterium Microcystis aeruginosa.

This investigation examines how extracellular polymeric substances (EPSs) and environmental factors affect the bioaccumulation and toxicity of inorganic mercury (+2 oxidation state, Hg(II)) using a culture of Microcystis aeruginosa, which dominates eutrophic reservoir populations. The identified EPSs were classified as carbohydrates and proteins. Evaluation of the bioaccumulation of Hg(II) in cells by multiple regression analysis reveals that the concentration of EPSs in filtrate, the initial concentration of Hg(II) in medium, and the culture age significantly affected the amount of Hg(II) accumulated. Composition profiles revealed that the concentrations of soluble carbohydrates were significantly higher in Hg(II)-accumulated cells than in the control ones. Preliminary results based on scanning electron microscopic (SEM) map investigations suggest that most of the Hg(II) was accumulated in the cytoplasm (intracellular). Additionally, the effective concentrations (EC50) of Hg(II) that inhibit the growth of M. aeruginosa were 38.6 µg L(-1) in the logarithmic phase and 17.5 µg L(-1) in the stationary phase. As expected, the production of more EPSs in the logarithmic phase typically implies higher EC50 values because EPSs may be regarded as a protective barrier of cells against an external Hg(II) load, enabling them to be less influenced by Hg(II).

Type three secretion system-mediated escape of Burkholderia pseudomallei into the host cytosol is critical for the activation of NFκB.

BACKGROUND: Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic in Southeast Asia and Northern Australia. This Gram-negative pathogen possesses numerous virulence factors including three "injection type" type three secretion systems (T3SSs). B. pseudomallei has been shown to activate NFκB in HEK293T cells in a Toll-like receptor and MyD88 independent manner that requires T3SS gene cluster 3 (T3SS3 or T3SSBsa). However, the mechanism of how T3SS3 contributes to NFκB activation is unknown. RESULTS: Known T3SS3 effectors are not responsible for NFκB activation. Furthermore, T3SS3-null mutants are able to activate NFκB almost to the same extent as wildtype bacteria at late time points of infection, corresponding to delayed escape into the cytosol. NFκB activation also occurs when bacteria are delivered directly into the cytosol by photothermal nanoblade injection. CONCLUSIONS: T3SS3 does not directly activate NFκB but facilitates bacterial escape into the cytosol where the host is able to sense the presence of the pathogen through cytosolic sensors leading to NFκB activation.

Electrical impedance monitoring of photothermal porated mammalian cells.

To transfer large cargo into mammalian cells, we recently provided a new approach called a photothermal nanoblade. Micron-sized membrane pores generated by the nanoblade are surprisingly well repaired with little cell death, suggesting rapid membrane-resealing dynamics. Here, we report the resealing time of photothermal porated mammalian cell plasma membranes using an electrical impedance sensor. Cell membrane pores were generated by high-speed cavitation bubbles induced by laser pulsing of metallic microdisks on a pair of transparent indium tin oxide electrodes. Electrical responses from the sensor electrodes were obtained with a signal voltage of 500 mV and a frequency at 500 kHz. Real-time impedance measurements show that membrane resealing and impedance recovery take a surprisingly long 1 to 2 min after laser pulsing. A nonrecovering impedance shift is also detected for cells after high-energy laser pulsing. This impedance response is also confirmed by a separate experiment in which thin-film gold electrodes are used to trigger cavitation bubbles for opening transient membrane pores on cells cultured on electrodes. Overall, our study platform provides new insight for micron-sized membrane defect repair dynamics to maintain cell viability.

3D pulsed laser-triggered high-speed microfluidic fluorescence-activated cell sorter.

We report a 3D microfluidic pulsed laser-triggered fluorescence-activated cell sorter capable of sorting at a throughput of 23 000 cells per s with 90% purity in high-purity mode and at a throughput of 45 000 cells per s with 45% purity in enrichment mode in one stage and in a single channel. This performance is realized by exciting laser-induced cavitation bubbles in a 3D PDMS microfluidic channel to generate high-speed liquid jets that deflect detected fluorescent cells and particles focused by 3D sheath flows. The ultrafast switching mechanism (20 µs complete on-off cycle), small liquid jet perturbation volume, and three-dimensional sheath flow focusing for accurate timing control of fast (1.5 m s(-1)) passing cells and particles are three critical factors enabling high-purity sorting at high-throughput in this sorter.

Detection of mRNA in living cells by double-stranded locked nucleic acid probes.

Double-stranded probes are homogeneous biosensors for rapid detection of specific nucleotide sequences. These double-stranded probes have been applied in various molecular sensing applications, such as real-time polymerase chain reaction and detection of bacterial 16S rRNA. In this study, we present the design and optimization of double-stranded probes for single-cell gene expression analysis in living cells. With alternating DNA/LNA monomers for optimizing the stability and specificity, we show that the probe is stable in living cells for over 72 hours post-transfection and is capable of detecting changes in gene expression induced by external stimuli. The probes can be delivered to a large number of cells simultaneously by cationic liposomal transfection or to individual cells selectively by photothermal delivery. We also demonstrate that the probe quantifies intracellular mRNA in living cells through the use of an equilibrium analysis. With its effectiveness and performance, the double-stranded probe represents a broadly applicable approach for large-scale single-cell gene expression analysis toward numerous biomedical applications, such as systems biology, cancer, and drug screening.

Nanoblade delivery and incorporation of quantum dot conjugates into tubulin networks in live cells.

Quantum dots (QDs) have not been used to label cytoskeleton structure of live cells owing to limitations in delivery strategies, and QDs conjugation methods and issues with nonspecific binding. We conjugated tubulin to QDs and applied the emerging method of photothermal nanoblade to deliver QD-tubulin conjugates into live Hela cells. This method will open new opportunities for cytosolic targeting of QDs in live cells.

Scanning laser pulses driven microfluidic peristaltic membrane pump.

We reported a pulsed laser driven peristaltic pump for driving fluid in multilayer polydimethylsiloxane (PDMS) microchannels. By synchronizing the dynamics of deforming membrane valves with pulsed laser generated bubbles, a maximum pumping rate of 460 pl s(-1) has been achieved.

Pulsed laser triggered high speed microfluidic fluorescence activated cell sorter.

We report a high speed and high purity pulsed laser triggered fluorescence activated cell sorter (PLACS) with a sorting throughput up to 20,000 mammalian cells s(-1) with 37% sorting purity, 90% cell viability in enrichment mode, and >90% purity in high purity mode at 1500 cells s(-1) or 3000 beads s(-1). Fast switching (30 µs) and a small perturbation volume (~90 pL) is achieved by a unique sorting mechanism in which explosive vapor bubbles are generated using focused laser pulses in a single layer microfluidic PDMS channel.

Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade.

Burkholderia pseudomallei and Burkholderia thailandensis are related pathogens that invade a variety of cell types, replicate in the cytoplasm, and spread to nearby cells. We have investigated temporal and spatial requirements for virulence determinants in the intracellular life cycle, using genetic dissection and photothermal nanoblade delivery, which allows efficient placement of bacterium-sized cargo into the cytoplasm of mammalian cells. The conserved Bsa type III secretion system (T3SS(Bsa)) is dispensable for invasion, but is essential for escape from primary endosomes. By nanoblade delivery of B. thailandensis we demonstrate that all subsequent events in intercellular spread occur independently of T3SS(Bsa) activity. Although intracellular movement was essential for cell-cell spread by B. pseudomallei and B. thailandensis, neither BimA-mediated actin polymerization nor the formation of membrane protrusions containing bacteria was required for B. thailandensis. Surprisingly, the cryptic (fla2) flagellar system encoded on chromosome 2 of B. thailandensis supported rapid intracellular motility and efficient cell-cell spread. Plaque formation by both pathogens was dependent on the activity of a type VI secretion system (T6SS-1) that functions downstream from T3SS(Bsa)-mediated endosome escape. A remarkable feature of Burkholderia is their ability to induce the formation of multinucleate giant cells (MNGCs) in multiple cell types. By infection and nanoblade delivery, we observed complete correspondence between mutant phenotypes in assays for cell fusion and plaque formation, and time-course studies showed that plaque formation represents MNGC death. Our data suggest that the primary means for intercellular spread involves cell fusion, as opposed to pseudopod engulfment and bacterial escape from double-membrane vacuoles.

High-speed droplet generation on demand driven by pulse laser-induced cavitation.

We report on a pulse laser-driven droplet generation (PLDG) mechanism that enables on-demand droplet generation at rates up to 10,000 droplets per second in a single-layer PDMS-based microfluidic device. Injected droplet volumes can be continuously tuned between 1 pL and 150 pL with less than 1% volume variation.

Photothermal nanoblade for large cargo delivery into mammalian cells.

It is difficult to achieve controlled cutting of elastic, mechanically fragile, and rapidly resealing mammalian cell membranes. Here, we report a photothermal nanoblade that utilizes a metallic nanostructure to harvest short laser pulse energy and convert it into a highly localized explosive vapor bubble, which rapidly punctures a lightly contacting cell membrane via high-speed fluidic flows and induced transient shear stress. The cavitation bubble pattern is controlled by the metallic structure configuration and laser pulse duration and energy. Integration of the metallic nanostructure with a micropipet, the nanoblade generates a micrometer-sized membrane access port for delivering highly concentrated cargo (5 × 10(8) live bacteria/mL) with high efficiency (46%) and cell viability (>90%) into mammalian cells. Additional biologic and inanimate cargo over 3-orders of magnitude in size including DNA, RNA, 200 nm polystyrene beads, to 2 µm bacteria have also been delivered into multiple mammalian cell types. Overall, the photothermal nanoblade is a new approach for delivering difficult cargo into mammalian cells.

Photothermal nanoblade for patterned cell membrane cutting.

We report a photothermal nanoblade that utilizes a metallic nanostructure to harvest short laser pulse energy and convert it into a highly localized and specifically shaped explosive vapor bubble. Rapid bubble expansion and collapse punctures a lightly-contacting cell membrane via high-speed fluidic flows and induced transient shear stress. The membrane cutting pattern is controlled by the metallic nanostructure configuration, laser pulse polarization, and energy. Highly controllable, sub-micron sized circular hole pairs to half moon-like, or cat-door shaped, membrane cuts were realized in glutaraldehyde treated HeLa cells.