Cell Biology

Centrosomes are dynamic organelles critical for mitotic spindle assembly and cilia formation. Here, I describe a protocol for quantifying relative centrosomal protein abundance in Drosophila melanogaster embryos using radial profile analysis of fluorescence intensity. The method involves embryo collection, manual dechorionation, mounting for live imaging, confocal microscopy, and subsequent image analysis. Radial profiling allows quantification of relative protein abundance together with its spatial distribution at the centrosome, providing either relative or normalized intensity profiles. I then outline how this approach can be integrated with complementary techniques such as fluorescence recovery after photobleaching (FRAP) and super-resolution imaging, in this case, three-dimensional structured illumination microscopy (3D-SIM). Combining radial fluorescence profiling with these imaging modalities enables high-resolution, quantitative analysis of dynamic centrosome assembly in a genetically tractable system.

ER-phagy, a selective autophagy process crucial for maintaining cellular homeostasis by targeting the endoplasmic reticulum (ER), has been challenging to study in vivo due to the lack of suitable spatiotemporal quantification tools. Existing methods like electron microscopy, biochemical assays, and in vitro reporters lack resolution, scalability, or physiological relevance. Here, we present a detailed protocol for generating two transgenic mouse models: ER-TRG (constitutively expressing an ER lumen-targeting tandem RFP-GFP tag) and CA-ER-TRG (Cre-recombinase-activated ER-TRG). Additionally, we outline procedures for quantitative imaging of ER-phagy in vivo, covering tissue preparation, confocal microscopy, and signal analysis. This protocol offers a robust and reproducible tool for investigating ER-phagy dynamics across various tissues, developmental stages, and pathophysiological conditions, facilitating both fundamental and translational research.

Protein synthesis and degradation (i.e., turnover) forms an important part of protein homeostasis and has been implicated in many age-associated diseases. Different cellular locations, such as organelles and membraneless compartments, often contain individual protein quality control and degradation machineries. Conventional methods to assess protein turnover across subcellular compartments require targeted genetic manipulation or isolation of specific organelles. Here we describe a protocol for simultaneous proteome localization and turnover (SPLAT) analysis, which combines protein turnover measurements with unbiased subcellular spatial proteomics to measure compartment-specific protein turnover rates on a proteome-wide scale. This protocol utilizes dynamic stable isotope labeling of amino acids in cell culture (dynamic SILAC) to resolve the temporal information of protein turnover and multi-step differential ultracentrifugation to assign proteins to multiple subcellular localizations. We further incorporate 2D liquid chromatography fractionation to greatly increase analytical depth while multiplexing with tandem mass tags (TMT) to reduce acquisition time 10-fold. This protocol resolves the spatial and temporal distributions of proteins and can also reveal temporally distinct spatial localizations within a protein pool.

Phosphoinositides are rare membrane lipids that mediate cell signaling and membrane dynamics. PI(4)P and PI(3)P are two major phosphoinositides crucial for endolysosomal functions and dynamics, making them the lipids of interest in many studies. The acute modulation of phosphoinositides at a given organelle membrane can reveal important insights into their cellular function. Indeed, the localized depletion of PI(4)P and PI(3)P is a viable tool to assess the importance of these phosphoinositides in various experimental conditions. Here, we describe a live imaging method to acutely deplete PI(4)P and PI(3)P on endolysosomes. The depletion assay utilizes the GAI-GID1 or the FRB-FKBP inducible dimerization system to target the catalytic domain of the PI(4)P phosphatase, Sac1, or the PI(3)P phosphatase domain of MTM1 to the endolysosome for localized depletion of these phosphoinositides. By using the fluorescently tagged biosensors, 2xP4M and PX, we can validate and monitor the depletion of PI(4)P and PI(3)P, respectively, on endolysosomes in real-time. We discuss a method for normalizing the fluorescence measurements to appropriate the relative amount of these phosphoinositides in the organellar membranes (endolysosomes), which is required for monitoring PI(4)P or PI(3)P levels during the acute depletion assay. Since the localization of the dimerization partners is specified by the membrane targeting signal, our protocol will be useful for studying the signaling and functions of phosphoinositides at any membrane.

Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples.

Key features

• Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET).

• Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms.

• The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details.

Graphical overview

Mammalian orthoreoviruses (reoviruses) are nonenveloped, double-stranded RNA viruses that replicate and assemble in cytoplasmic membranous organelles called viral inclusions (VIs). To define the cellular compartments involved in nonlytic reovirus egress, we imaged viral egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs). Electron and confocal microscopy showed that reovirus mature virions are recruited from VIs to modified lysosomes termed sorting organelles (SOs). Later in infection, membranous carriers (MCs) emerge from SOs and transport new virions to the plasma membrane for nonlytic egress. Transmission electron microscopy (TEM) combined with electron tomography (ET) and three-dimensional (3D) reconstruction revealed that these compartments are connected and form the exit pathway. Connections are established by channels through which mature virions are transported from VIs to MCs. In the last step, MCs travel across the cytoplasm and fuse with the plasma membrane, which facilitates reovirus egress. This bio-protocol describes the combination of imaging approaches (TEM, ET, and 3D reconstruction) to analyze reovirus egress zones. The spatial information present in the 3D reconstructions, along with the higher resolution relative to 2D projections, allowed us to identify components of a new nonlytic viral egress pathway.

Over the years, studying the ultrastructure of the eukaryotic cilia/flagella using electron microscopy (EM) has contributed significantly toward our understanding of ciliary function. Major complexes in the cilia, such as inner and outer dynein arms, radial spokes, and dynein regulatory complexes, were originally discovered by EM. Classical resin-embedding EM or cryo-electron tomography can be performed directly on the isolated cilia or in some cases, cilia directly attached to the cell body. Recently, single particle cryo-EM has emerged as a powerful structural technique to elucidate high-resolution structures of macromolecular complexes; however, single particle cryo-EM requires non-overlapping complexes, i.e., the doublet microtubule of the cilia. Here, we present a protocol to separate the doublet microtubule from the isolated cilia bundle of two species, Tetrahymena thermophila and Chlamydomonas reinhardtii, using ATP reactivation and sonication. Our approach produces good distribution and random orientation of the doublet microtubule fragments, which is suitable for single particle cryo-EM analysis.