Automated Quantification of Subcellular Particles in Myogenic Progenitors.

Fluorescence microscopy is a powerful tool enabling the visualization of protein localization within cells. In this article, we outline an automated and non-biased way to detect and quantify subcellular particles using immunocytochemistry, fluorescence microscopy, and the program CellProfiler. We discuss the examination of two types of subcellular particles: messenger ribonucleoprotein (mRNP) granules, namely processing bodies and stress granules, and autophagosomes. Fluorescent microscopy Z-stacks are acquired and deconvolved, and maximum intensity images are generated. The number of subcellular particles per cell is then quantified using the described CellProfiler pipeline. We also explain how to isolate primary myoblast progenitor cells from mice, which were used to obtain the presented results. Last, we discuss the critical parameters to be considered for each of these techniques. Both mRNP granules and autophagosomes play important roles in sequestering intracellular cargo, such as messenger RNAs and RNA-binding proteins for mRNP granules and cytoplasmic waste for autophagosomes. The methods outlined in this article are widely applicable for studies relating to subcellular particle formation, localization, and flux during homeostasis, following stimuli, and during disease. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescence microscopy of messenger ribonucleoprotein granules in primary myoblasts Alternate Protocol: Immunofluorescence microscopy of autophagosomes in primary myoblasts Support Protocol: Isolation of primary myoblasts from mice Basic Protocol 2: Automated quantification of subcellular particles.

Vesicle Dynamics during Plant Cell Cytokinesis Reveals Distinct Developmental Phases.

Cell division in plant cells requires the deposition of a new cell wall between the two daughter cells. The assembly of this plate requires the coordinated movement of cargo vesicles whose size is below the diffraction-limited resolution of the optical microscope. We combined high spatial and temporal resolution confocal laser scanning microscopy with advanced image-processing tools and fluorescence fluctuation methods and distinguished three distinct phases during cell plate expansion in tobacco (Nicotiana tabacum) 'Bright Yellow-2' cells: massive delivery of preexisting vesicles to a disk-shaped region at the equatorial plane precedes a primary rapid expansion phase followed by a secondary, slow expansion phase during which the extremity of the circular plate seeks contact with the mother wall and brings about the separation of the two portions of cytoplasm. Different effects of pharmacological inhibition emphasize the distinct nature of the assembly and expansion mechanisms characterizing these phases.

The plasma membrane protein Rch1 is a negative regulator of cytosolic calcium homeostasis and positively regulated by the calcium/calcineurin signaling pathway in budding yeast.

Saccharomyces cerevisiae Rch1 is structurally similar to both the vertebrate solute carrier SLC10A7 and Candida albicans Rch1. We show here that ScRCH1 is a functional homolog of CaRCH1. In S. cerevisiae, overexpression of ScRCH1 suppresses, but deletion of ScRCH1 does not affect, the lithium and rapamycin tolerance of pmr1 cells. Overexpression of ScRCH1 reduces expression of ENA1, prevents sustained accumulation of cytosolic calcium and reduces the activation level of calcium/calcineurin signaling in pmr1 cells. Therefore, similar to the situation in the pathogen C. albicans, ScRch1 negatively regulates the cytosolic homeostasis in response to high levels of extracellular calcium. ScRch1 proteins distribute as multiple foci in the plasma membrane prior to cell division, move toward and concentrate at the bud neck as the bud grows in size, and disperse again along the plasma membrane immediately prior to cytokinesis. Furthermore, our genetic and biochemical data also demonstrate that transcriptional expression of RCH1 is positively regulated by calcium/calcineurin signaling through the sole CDRE element in its promoter.

Anillin interacts with microtubules and is part of the astral pathway that defines cortical domains.

Cytokinesis occurs by the ingression of an actomyosin ring that separates the cell into two daughter cells. The mitotic spindle, comprising astral and central spindle microtubules, couples contractile ring ingression with DNA segregation. Cues from the central spindle activate RhoA, the upstream regulator of the contractile ring. However, additional cues from the astral microtubules also reinforce the localization of active RhoA. Using human cells, we show that astral and central spindle microtubules independently control the localization of contractile proteins during cytokinesis. Astral microtubules restrict the accumulation and localization of contractile proteins during mitosis, whereas the central spindle forms a discrete ring by directing RhoA activation in the equatorial plane. Anillin stabilizes the contractile ring during cytokinesis. We show that human anillin interacts with astral microtubules and that this interaction is competed by the cortical recruitment of anillin by active RhoA. Anillin restricts the localization of myosin to the equatorial cortex and that of NuMA (part of the microtubule-tethering complex that regulates spindle position) to the polar cortex. The sequestration of anillin by astral microtubules might alter the organization of cortical proteins to polarize cells for cytokinesis.