Biochemistry

Mitochondria are vital organelles essential for cellular functions, but their lipid composition and response to stressors are not fully understood. Recent advancements in lipidomics reveal insights into lipid functions, especially their roles in metabolic perturbations and diseases. Previous methods have focused on the protein composition of mitochondria and mitochondrial-associated membranes. The advantage of our technique is that it combines organelle isolation with targeted lipidomics, offering new insights into the composition and dynamics of these organelles in pathological conditions. We developed a mitochondria isolation protocol for L6 myotubes, enabling lipidomics analysis of specific organelles without interference from other cellular compartments. This approach offers a unique opportunity to dissect lipid dynamics within mitochondria and their associated ER compartments under cellular stress.

Key features

• Analysis and quantification of lipids in mitochondria–ER fraction through liquid chromatography–tandem mass spectrometry-based lipidomics (LC-MS/MS lipidomics).

• LC-MS/MS lipidomics provide precise and unbiased information on the lipid composition in in vitro systems.

• LC-MS/MS lipidomics facilitates the identification of lipid signatures in mammalian cells.

Microglia, the brain's primary resident immune cell, exists in various phenotypic states depending on intrinsic and extrinsic signaling. Distinguishing between these phenotypes can offer valuable biological insights into neurodevelopmental and neurodegenerative processes. Recent advances in single-cell transcriptomic profiling have allowed for increased granularity and better separation of distinct microglial states. While techniques such as immunofluorescence and single-cell RNA sequencing (scRNA-seq) are available to differentiate microglial phenotypes and functions, these methods present notable limitations, including challenging quantification methods, high cost, and advanced analytical techniques. This protocol addresses these limitations by presenting an optimized cell preparation procedure that prevents ex vivo activation and a flow cytometry panel to distinguish four distinct microglial states from murine brain tissue. Following cell preparation, fluorescent antibodies were applied to label 1) homeostatic, 2) disease-associated (DAM), 3) interferon response (IRM), and 4) lipid-droplet accumulating (LDAM) microglia, based on gene markers identified in previous scRNA-Seq studies. Stained cells were analyzed by flow cytometry to assess phenotypic distribution as a function of age and sex. A key advantage of this procedure is its adaptability, allowing the panel provided to be enhanced using additional markers with an appropriate cell analyzer (i.e., Cytek Aurora 5 laser spectral flow cytometer) and interrogating different brain regions or disease models. Additionally, this protocol does not require microglial cell sorting, resulting in a relatively quick and straightforward experiment. Ultimately, this protocol can compare the distribution of microglial phenotypic states between various experimental groups, such as disease state or age, with a lower cost and higher throughput than scRNA-seq.

The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems—the super-sensitive AID and AID 2—were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker–based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)–dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts.

Agrobacterium-mediated transient gene expression in Nicotiana benthamiana is widely used to study gene function in plants. One dramatic phenotype that is frequently screened for is cell death. Here, we present a simplified protocol for Agrobacterium-mediated transient gene expression by infiltration. Compared with current methods, the novel protocol can be done without a centrifuge or spectrometer, thereby suitable for K-12 outreach programs as well as rapidly identifying genes that induce cell death.

The eye is a complex organ composed of multiple tissues in anterior and posterior eye segments. Malfunctions of any of these tissues can lead to ocular diseases and loss of vision. A detailed understanding of the ocular anatomy and physiology in animal models and humans contributes to the development of ocular drugs by enabling studies on drug delivery and clearance routes, pharmacokinetics, and toxicity. This protocol provides step-by-step instructions for the extraction and homogenization of ocular tissues for enzymatic and proteomics analyses.

The field of oligonucleotide therapeutics is rapidly advancing, particularly for combating orphan diseases and cancer. However, the intrinsic instability of oligonucleotides, especially RNA, poses a substantial challenge in the face of the harsh conditions encountered intracellularly and in circulation. Therefore, evaluating the stability of oligos in serum is of great significance when developing oligonucleotide therapeutics. This protocol outlines a dependable and reproducible method for preparing oligonucleotide duplexes, coupled with confirmation by gel electrophoresis. Subsequently, the protocol defines a mechanism to assess the stability of the oligo duplexes in serum. This protocol seeks to establish a standardized reference for researchers, enabling them to compare the impact of various modifications on oligo stability and assess the degradation kinetics effectively.

The assessment of peptide–protein interactions is a pivotal aspect of studying the functionality and mechanisms of various bioactive peptides. In this context, it is essential to employ methods that meet specific criteria, including sensitivity, biocompatibility, versatility, simplicity, and the ability to offer real-time monitoring. In cellular contexts, only a few proteins naturally possess inherent fluorescence, specifically those containing aromatic amino acids, particularly tryptophan. Nonetheless, by covalently attaching fluorescent markers, almost all proteins can be modified for monitoring purposes. Among the early extrinsic fluorescent probes designed for this task, dansyl chloride (DNSC) is a notable option due to its versatile nature and reliable performance. DNSC has been the primary choice as a fluorogenic derivatizing reagent for analyzing amino acids in proteins and peptides for an extended period of time. In our work, we have effectively utilized the distinctive properties of dansylated-calmodulin (D-CaM) for monitoring the interaction dynamics between proteins and peptides, particularly in the context of their association with calmodulin (CaM), a calcium-dependent regulatory protein. This technique not only enables us to scrutinize the affinity of diverse ligands but also sheds light on the intricate role played by calcium in these interactions.

Key features

• Dynamic fluorescence and real-time monitoring: dansyl-modified CaM enables sensitive, real-time fluorescence, providing valuable insights into the dynamics of molecular interactions and ligand binding.

• Selective interaction and stable fluorescent adducts: DNSC selectively interacts with primary amino groups, ensuring specific detection and forming stable fluorescent sulfonamide adducts.

• Versatility in research and ease of identification: D-CaM is a versatile tool in biological research, facilitating identification, precise quantification, and drug assessment for therapeutic development.

• Sensitivity to surrounding alterations: D-CaM exhibits sensitivity to its surroundings, particularly ligand-induced changes, offering subtle insights into molecular interactions and environmental influences.

Graphical overview

Fluorescence emission profiles of dansylated-calmodulin (D-CaM) in different states. Fluorescence emission spectra of D-CaM upon excitation at 320 nm are depicted. Conditions include apo-D-CaM (gray), holo-D-CaM (red), apo-D-CaM bound to peptide (blue), and holo-D-CaM bound to peptide (purple). Corresponding structural representations of D-CaM next to each condition are superimposed on the respective spectra along with the hydrophobicity of the dansyl environment, which increases upon binding of peptide or Ca²⁺ to D-CaM. Upon peptide binding to D-CaM, there is an enhancement in the fluorescent intensity of the spectra; upon Ca²⁺ binding, there is an enhancement of the intensity and a leftward shift of the spectra.

Contractile injection systems (CISs), one of the most important bacterial secretion systems that transport substrates across the membrane, are a collection of diverse but evolutionarily related macromolecular devices. Numerous effector proteins can be loaded and injected by this secretion complex to their specific destinations. One group of CISs called extracellular CIS (eCIS) has been proposed as secretory molecules that can be released from the bacterial cytoplasm and attack neighboring target cells from the extracellular environment. This makes them a potential delivery vector for the transportation of various cargos without the inclusion of bacterial cells, which might elicit certain immunological responses from hosts. We have demonstrated that the Photorhabdus virulence cassette (PVC), which is a typical eCIS, could be applied as an ideal vector for the translocation of proteinaceous cargos with different physical or chemical properties. Here, we describe the in-depth purification protocol of this mega complex from Escherichia coli. The protocol provided is a simpler, faster, and more productive way of generating the eCIS complexes than available methodologies reported previously, which can facilitate the subsequent applications of these nanodevices and other eCIS in different backgrounds.

The polymerase chain reaction (PCR) is an extensively used technique to quickly and accurately make many copies of a specific segment of DNA. In addition to naturally existing DNA polymerases, PCR utilizes a range of genetically modified recombinant DNA polymerases, each characterized by varying levels of processivity and fidelity. Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA-binding protein, with Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity but is sold at a sumptuously high price under various trade names and commercial variants. We recently reported a quick and easy purification protocol that utilizes ethanol or acetone to precipitate Pfu-Sso7d from heat-cleared lysates. We also optimized a PCR buffer solution that outperforms commercial buffers when used with Pfu-Sso7d. Here, we provide a step-by-step guide on how to purify recombinant Pfu-Sso7d. This purification protocol and the buffer system will offer researchers cost-efficient access to fusion polymerase.

Key features

• We detail a precipitation-based protocol utilizing ethanol and acetone for purifying Pfu-Sso7d.

• Despite ethanol and acetone displaying effective precipitation efficiency, acetone is preferred for its superior performance.

• Furthermore, we present a PCR buffer that outperforms commercially available PCR buffers.

• The Pfu-Sso7d purified in-house and the described PCR buffer exhibit excellent performance in PCR applications.

Candida glabrata is an opportunistic pathogen that may cause serious infections in an immunocompromised host. C. glabrata cell wall proteases directly interact with host cells and affect yeast virulence and host immune responses. This protocol describes methods to purify β-1,3-glucan-bonded cell wall proteases from C. glabrata. These cell wall proteases are detached from the cell wall glucan network by lyticase treatment, which hydrolyzes β-1,3-glucan bonds specifically without rupturing cells. The cell wall supernatant is further fractioned by centrifugal devices with cut-offs of 10 and 50 kDa, ion-exchange filtration(charge), and gel filtration (size exclusion). The enzymatic activity of C. glabrata proteases is verified with MDPF-gelatin zymography and the degradation of gelatin is visualized by loss of gelatin fluorescence. With this procedure, the enzymatic activities of the fractions are kept intact, differing from methods used in previous studies with trypsin digestion of the yeast cell wall. The protein bands may be eventually located from a parallel silver-stained gel and identified with LC–MS/MS spectrometry. The advantage of this methodology is that it allows further host protein degradation assays; the protocol is also suitable for studying other Candida yeast species.

Key features

• Uses basic materials and laboratory equipment, enabling low-cost studies.

• Facilitates the selection and identification of proteases with certain molecular weights.

• Enables further functional studies with host proteins, such as structural or immune response–related, or enzymes and candidate protease inhibitors(e.g., from natural substances).

• This protocol has been optimized for C. glabrata but may be applied with modifications to other Candida species.

Graphical overview