Fluorescence microscopy has become an essential tool in cell biology. This technique allows researchers to visualize the dynamics of tissue, cells, individual organelles, and macromolecular ...assemblies inside the cell. Unfortunately, fluorescence microscopy is not completely "noninvasive" as the high-intensity excitation light required for excitation of fluorophores is inherently toxic for live cells. Physiological changes induced by excessive illumination can lead to artifacts and abnormal responses. In this chapter, we review major factors that contribute to phototoxicity and discuss practical solutions for circumventing photodamage. These solutions include the proper choice of image acquisition parameters, optimization of filter sets, hardware synchronization, and the use of intelligent illumination to avoid unnecessary light exposure.
The shape of walled cells such as fungi, bacteria, and plants are determined by the cell wall. Models for cell morphogenesis postulate that the effects of turgor pressure and mechanical properties of ...the cell wall can explain the shapes of these diverse cell types 1–6. However, in general, these models await validation through quantitative experiments. Fission yeast Schizosaccharomyces pombe are rod-shaped cells that grow by tip extension and then divide medially through formation of a cell wall septum. Upon cell separation after cytokinesis, the new cell ends adopt a rounded morphology. Here, we show that this shape is generated by a very simple mechanical-based mechanism in which turgor pressure inflates the elastic cell wall in the absence of cell growth. This process is independent of actin and new cell wall synthesis. To model this morphological change, we first estimate the mechanical properties of the cell wall using several approaches. The lateral cell wall behaves as an isotropic elastic material with a Young’s modulus of 50 ± 10 MPa inflated by a turgor pressure estimated to be 1.5 ± 0.2 MPa. Based upon these parameters, we develop a quantitative mechanical-based model for new end formation that reveals that the cell wall at the new end expands into its characteristic rounded shape in part because it is softer than the mature lateral wall. These studies provide a simple example of how turgor pressure expands the elastic cell wall to generate a particular cell shape.
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•Mechanical properties of the S. pombe cell wall and turgor pressure are measured•Formation of the rounded new cell end is independent of actin and growth•Turgor pressure mechanically deforms the flat septum cell wall to a rounded shape•Simulations of bulging reveal mechanical properties of the septum cell wall
Shapes of walled cells are determined by mechanical properties of the cell wall and turgor pressure. After rod-shaped S. pombe cells divide, the cell wall at the new end of the cell adopts a rounded shape. Here, Atilgan et al. show that this shape is generated by turgor pressure simply inflating the elastic cell wall in the absence of cell growth.
Defining mechanisms that maintain tissue stem cells during homeostasis, stress, and aging is important for improving tissue regeneration and repair and enhancing cancer therapies. Here, we show that ...Id1 is induced in hematopoietic stem cells (HSCs) by cytokines that promote HSC proliferation and differentiation, suggesting that it functions in stress hematopoiesis. Genetic ablation of Id1 increases HSC self-renewal in serial bone marrow transplantation (BMT) assays, correlating with decreases in HSC proliferation, mitochondrial biogenesis, and reactive oxygen species (ROS) production. Id1−/− HSCs have a quiescent molecular signature and harbor less DNA damage than control HSCs. Cytokines produced in the hematopoietic microenvironment after γ-irradiation induce Id1 expression. Id1−/− HSCs display a blunted proliferative response to such cytokines and other inducers of chronic proliferation including genotoxic and inflammatory stress and aging, protecting them from chronic stress and exhaustion. Thus, targeting Id1 may be therapeutically useful for improving HSC survival and function during BMT, chronic stress, and aging.
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•Id1−/− HSCs have enhanced self-renewal potential and are maintained during BMT•Id1−/− HSCs show reduced oxidative stress and increased quiescence after BMT•Id1−/− HSCs are protected from inflammatory cytokine-induced proliferative stress•Id1−/− HSCs are protected from exhaustion by chronic inflammatory stress and aging
Singh et al. show that Id1 is an important mediator of stress hematopoiesis. They found that Id1 deletion protects HSCs from exhaustion in multiple paradigms of chronic and physiologically relevant stress and promotes their quiescence, suggesting that targeting Id1 may improve HSC survival and function during chronic stress and aging.
Centrosome overduplication promotes mitotic abnormalities, invasion and tumorigenesis. Cells regulate the number of centrosomes by limiting centriole duplication to once per cell cycle. The ...orthogonal orientation between a mother and a daughter centriole, established at the time of centriole duplication, is thought to block further duplication of the mother centriole. Loss of orthogonal orientation (disengagement) between two centrioles during anaphase is considered a licensing event for the next round of centriole duplication. Disengagement requires the activity of Polo-like kinase 1 (Plk1), but how Plk1 drives this process is not clear. Here we employ correlative live/electron microscopy and demonstrate that Plk1 induces maturation and distancing of the daughter centriole, allowing reduplication of the mother centriole even if the original daughter centriole is still orthogonal to it. We find that mother centrioles can undergo reduplication when original daughter centrioles are only ∼80 nm apart, which is the distance centrioles normally reach during prophase.
Far-red cyanine fluorophores find extensive use in modern microscopy despite modest quantum yields. To improve the photon output of these molecules, we report a synthetic strategy that blocks the ...major deactivation pathway: excited-state trans-to-cis polyene rotation. In the key transformation, a protected dialdehyde precursor undergoes a cascade reaction to install the requisite tetracyclic ring system. The resulting molecules exhibit the characteristic features of conformational restraint, including improved fluorescence quantum yield and extended lifetime. Moreover, these compounds recover from hydride reduction with dramatically improved efficiency. These observations enable efficient single-molecule localization microscopy in oxygenated buffer without addition of thiols. Enabled by modern organic synthesis, these studies provide a new class of far-red dyes with promising spectroscopic and chemical properties.
Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during ...spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize complete 3D movements of individual kinetochores throughout mitosis in nontransformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.
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► In human cells, chromosomes form a ring around the spindle during prometaphase ► This arrangement requires chromokinesin-mediated ejection of chromosome arms ► Formation of the ring accelerates spindle assembly ► Stable amphitelic attachments form during late prometaphase and metaphase
The intracellular ciliogenesis pathway requires membrane trafficking, fusion, and reorganization. Here, we demonstrate in human cells and zebrafish that the F-BAR domain containing proteins PACSIN1 ...and -2 play an essential role in ciliogenesis, similar to their binding partner and membrane reorganizer EHD1. In mature cilia, PACSINs and EHDs are dynamically localized to the ciliary pocket membrane (CPM) and transported away from this structure on membrane tubules along with proteins that exit the cilium. PACSINs function early in ciliogenesis at the ciliary vesicle (CV) stage to promote mother centriole to basal body transition. Remarkably, we show that PACSIN1 and EHD1 assemble membrane t7ubules from the developing intracellular cilium that attach to the plasma membrane, creating an extracellular membrane channel (EMC) to the outside of the cell. Together, our work uncovers a function for F-BAR proteins and membrane tubulation in ciliogenesis and explains how the intracellular cilium emerges from the cell.
During vertebrate development, the presomitic mesoderm (PSM) periodically segments into somites, which will form the segmented vertebral column and associated muscle, connective tissue, and dermis. ...The periodicity of somitogenesis is regulated by a segmentation clock of oscillating Notch activity. Here, we examined mouse mutants lacking only
or
, which we previously demonstrated act redundantly to prevent PSM differentiation.
is not required for somitogenesis, but
mutants display a range of vertebral defects. We analyzed
mutants by quantifying mRNAs fluorescently labeled by hybridization chain reaction within Imaris-based volumetric tissue subsets. These data indicate that FGF4 maintains
levels and normal oscillatory patterns. To support our hypothesis that FGF4 regulates somitogenesis through
, we demonstrate genetic synergy between
and
, but not with
. Our data indicate that
is potentially important in a spectrum of human Segmentation Defects of the Vertebrae caused by defective Notch oscillations.
Controlling the number of its centrioles is vital for the cell, as supernumerary centrioles cause multipolar mitosis and genomic instability. Normally, one daughter centriole forms on each mature ...(mother) centriole; however, a mother centriole can produce multiple daughters within a single cell cycle. The mechanisms that prevent centriole 'overduplication' are poorly understood. Here we use laser microsurgery to test the hypothesis that attachment of the daughter centriole to the wall of the mother inhibits formation of additional daughters. We show that physical removal of the daughter induces reduplication of the mother in S-phase-arrested cells. Under conditions when multiple daughters form simultaneously on a single mother, all of these daughters must be removed to induce reduplication. The number of daughter centrioles that form during reduplication does not always match the number of ablated daughter centrioles. We also find that exaggeration of the pericentriolar material (PCM) by overexpression of the PCM protein pericentrin in S-phase-arrested CHO cells induces formation of numerous daughter centrioles. We propose that that the size of the PCM cloud associated with the mother centriole restricts the number of daughters that can form simultaneously.
For proper segregation during cell division, each chromosome must connect to the poles of the spindle via microtubule bundles termed kinetochore fibers (K-fibers). K-fibers form by two distinct ...mechanisms: (1) capture of astral microtubules nucleated at the centrosome by the chromosomes' kinetochores or (2) attachment of kinetochores to noncentrosomal microtubules with subsequent transport of the minus ends of these microtubules toward the spindle poles. The relative contributions of these alternative mechanisms to normal spindle assembly remain unknown. In this study, we report that most kinetochores in human cells develop K-fibers via the second mechanism. Correlative light electron microscopy demonstrates that from the onset of spindle assembly, short randomly oriented noncentrosomal microtubules appear in the immediate vicinity of the kinetochores. Initially, these microtubules interact with the kinetochores laterally, but end-on attachments form rapidly in the first 3 min of prometaphase. Conversion from lateral to end-on interactions is impeded upon inhibition of the plus end-directed kinetochore-associated kinesin CenpE.
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