Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models.

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscope for reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

Unattached kinetochores rather than intrakinetochore tension arrest mitosis in taxol-treated cells.

Kinetochores attach chromosomes to the spindle microtubules and signal the spindle assembly checkpoint to delay mitotic exit until all chromosomes are attached. Light microscopy approaches aimed to indirectly determine distances between various proteins within the kinetochore (termed Delta) suggest that kinetochores become stretched by spindle forces and compact elastically when the force is suppressed. Low Delta is believed to arrest mitotic progression in taxol-treated cells. However, the structural basis of Delta remains unknown. By integrating same-kinetochore light microscopy and electron microscopy, we demonstrate that the value of Delta is affected by the variability in the shape and size of outer kinetochore domains. The outer kinetochore compacts when spindle forces are maximal during metaphase. When the forces are weakened by taxol treatment, the outer kinetochore expands radially and some kinetochores completely lose microtubule attachment, a condition known to arrest mitotic progression. These observations offer an alternative interpretation of intrakinetochore tension and question whether Delta plays a direct role in the control of mitotic progression.

Adaptive changes in the kinetochore architecture facilitate proper spindle assembly.

Mitotic spindle formation relies on the stochastic capture of microtubules at kinetochores. Kinetochore architecture affects the efficiency and fidelity of this process with large kinetochores expected to accelerate assembly at the expense of accuracy, and smaller kinetochores to suppress errors at the expense of efficiency. We demonstrate that on mitotic entry, kinetochores in cultured human cells form large crescents that subsequently compact into discrete structures on opposite sides of the centromere. This compaction occurs only after the formation of end-on microtubule attachments. Live-cell microscopy reveals that centromere rotation mediated by lateral kinetochore-microtubule interactions precedes the formation of end-on attachments and kinetochore compaction. Computational analyses of kinetochore expansion-compaction in the context of lateral interactions correctly predict experimentally observed spindle assembly times with reasonable error rates. The computational model suggests that larger kinetochores reduce both errors and assembly times, which can explain the robustness of spindle assembly and the functional significance of enlarged kinetochores.

Gender and the experience of moral distress in critical care nurses.

BACKGROUND: Nursing practice is complex, as nurses are challenged by increasingly intricate moral and ethical judgments. Inadequately studied in underrepresented groups in nursing, moral distress is a serious problem internationally for healthcare professionals with deleterious effects to patients, nurses, and organizations. Moral distress among nurses has been shown to contribute to decreased job satisfaction and increased turnover, withdrawal from patients, physical and psychological symptoms, and intent to leave current position or to leave the profession altogether. RESEARCH QUESTION: Do significant gender differences exist in the moral distress scores of critical care nurses? RESEARCH DESIGN: This study utilized a quantitative, descriptive methodology to explore moral distress levels in a sample of critical care nurses to determine whether gender differences exist in their mean moral distress scores. PARTICIPANTS AND RESEARCH CONTEXT: Participants (n = 31) were critical care nurses from an American Internet nursing community who completed the Moral Distress Scale-Revised online over a 5-day period in July 2013. ETHICAL CONSIDERATIONS: Institutional review board review approved the study, and accessing and completing the survey implied informed consent. FINDINGS: The results revealed a statistically significant gender difference in the mean moral distress scores of participants. Females reported statistically significantly higher moral distress scores than did males. Overall, the moral distress scores for both groups were relatively low. DISCUSSION: The findings of a gender difference have not previously been reported in the literature. However, other findings are consistent with previous studies on moral distress. CONCLUSION: Although the results of this study are not generalizable, they do suggest the need for continuing research on moral distress in underrepresented groups in nursing, including cultural and ethnic groups.

Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation.

Airway multiciliated epithelial cells play crucial roles in the mucosal defense system, but their differentiation process remains poorly understood. Mice lacking the basal body component Chibby (Cby) exhibit impaired mucociliary transport caused by defective ciliogenesis, resulting in chronic airway infection. In this paper, using primary cultures of mouse tracheal epithelial cells, we show that Cby facilitates basal body docking to the apical cell membrane through proper formation of ciliary vesicles at the distal appendage during the early stages of ciliogenesis. Cby is recruited to the distal appendages of centrioles via physical interaction with the distal appendage protein CEP164. Cby then associates with the membrane trafficking machinery component Rabin8, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Thus, our study identifies Cby as a key regulator of ciliary vesicle formation and basal body docking during the differentiation of airway ciliated cells.

Comparative and practical aspects of localization-based super-resolution imaging.

Super-resolution microscopy overcomes diffraction to generate images with superior resolution compared to conventional light microscopy. Localization-based super-resolution methods result in up to ten-fold improvement in resolution by determining the positions of fluorescent molecules with sub-pixel accuracy. This process critically depends on controlled emission at the level of individual fluorophores so that fluorescence is non-overlapping, allowing for accurate centroid determination of diffraction-limited spots by Gaussian fitting of the pixel intensities. The intrinsic photoswitching behavior of many fluorophores provides a convenient way to achieve emitter isolation. Here, we describe methods for label preparation and staining of cellular structures to obtain high-quality images using localization super resolution. We also compare labeling strategies and dye characteristics relevant to all localization-based techniques, such as STORM and PALM.

Kinetochore flexibility: creating a dynamic chromosome-spindle interface.

Kinetochores are complex macromolecular assemblies that link chromosomes to the mitotic spindle, mediate forces for chromosome motion, and generate the checkpoint signal delaying anaphase onset until all chromosomes are incorporated into the spindle. Proper execution of these functions depends on precise interactions between kinetochores and microtubules. While the molecular composition of the kinetochore is well described, structural organization of this organelle at the molecular and atomic levels is just beginning to emerge. Recent structural studies across scales suggest that kinetochores should not be viewed as rigid static scaffolds. Instead, these organelles exhibit a surprising degree of flexibility that enables rapid adaptations to various types of interactions with the mitotic spindle.

BubR1 is modified by sumoylation during mitotic progression.

BubR1 functions as a crucial component that monitors proper chromosome congression and mitotic timing during cell division. We investigated molecular regulation of BubR1 and found that BubR1 was modified by an unknown post-translation mechanism during the cell cycle, resulting in a significant mobility shift on denaturing gels. We termed it BubR1-M as the nature of modification was not characterized. Extended (>24 h) treatment of HeLa cells with a microtubule disrupting agent including nocodazole and taxol or release of mitotic shake-off cells into fresh medium induced BubR1-M. BubR1-M was derived from neither phosphorylation nor acetylation. Ectopic expression coupled with pulling down analyses showed that BubR1-M was derived from SUMO modification. Mutation analysis revealed that lysine 250 was a crucial site for sumoylation. Significantly, compared with the wild-type control, ectopic expression of a sumoylation-deficient mutant of BubR1 induced chromosomal missegregation and mitotic delay. Combined, our study identifies a new type of post-translational modification that is essential for BubR1 function during mitosis.

The spatial arrangement of chromosomes during prometaphase facilitates spindle assembly.

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.

Relative contributions of chromatin and kinetochores to mitotic spindle assembly.

During mitosis and meiosis in animal cells, chromosomes actively participate in spindle assembly by generating a gradient of Ran guanosine triphosphate (RanGTP). A high concentration of RanGTP promotes microtubule nucleation and stabilization in the vicinity of chromatin. However, the relative contributions of chromosome arms and centromeres/kinetochores in this process are not known. In this study, we address this issue using cells undergoing mitosis with unreplicated genomes (MUG). During MUG, chromatin is rapidly separated from the forming spindle, and both centrosomal and noncentrosomal spindle assembly pathways are active. MUG chromatin is coated with RCC1 and establishes a RanGTP gradient. However, a robust spindle forms around kinetochores/centromeres outside of the gradient peak. When stable kinetochore microtubule attachment is prevented by Nuf2 depletion in both MUG and normal mitosis, chromatin attracts astral microtubules but cannot induce spindle assembly. These results support a model in which kinetochores play the dominant role in the chromosome-mediated pathway of mitotic spindle assembly.

Chromosome congression in the absence of kinetochore fibres.

Proper chromosome congression (the process of aligning chromosomes on the spindle) contributes to accurate and faithful chromosome segregation. It is widely accepted that congression requires kinetochore fibres (K-fibres), microtubule bundles that extend from the kinetochores to spindle poles. Here, we demonstrate that chromosomes in human cells co-depleted of HSET (human kinesin-14) and hNuf2 (human Ndc80/Hec1-complex component) can congress to the metaphase plate in the absence of K-fibres. However, the chromosomes are not stably maintained at the metaphase plate under these conditions. Chromosome congression in HSET + hNuf2 co-depleted cells required the plus-end directed motor CENP-E (centromere protein E; kinesin-7 family member), which has been implicated in the gliding of mono-oriented kinetochores alongside adjacent K-fibres. Thus, proper end-on attachment of kinetochores to microtubules is not necessary for chromosome congression. Instead, our data support the idea that congression allows unattached chromosomes to move to the middle of the spindle where they have a higher probability of establishing connections with both spindle poles. These bi-oriented connections are also used to maintain stable chromosome alignment at the spindle equator.

The spindle assembly checkpoint is satisfied in the absence of interkinetochore tension during mitosis with unreplicated genomes.

The accuracy of chromosome segregation is enhanced by the spindle assembly checkpoint (SAC). The SAC is thought to monitor two distinct events: attachment of kinetochores to microtubules and the stretch of the centromere between the sister kinetochores that arises only when the chromosome becomes properly bioriented. We examined human cells undergoing mitosis with unreplicated genomes (MUG). Kinetochores in these cells are not paired, which implies that the centromere cannot be stretched; however, cells progress through mitosis. A SAC is present during MUG as cells arrest in response to nocodazole, taxol, or monastrol treatments. Mad2 is recruited to unattached MUG kinetochores and released upon their attachment. In contrast, BubR1 remains on attached kinetochores and exhibits a level of phosphorylation consistent with the inability of MUG spindles to establish normal levels of centromere tension. Thus, kinetochore attachment to microtubules is sufficient to satisfy the SAC even in the absence of interkinetochore tension.

Cooperative mechanisms of mitotic spindle formation.

Cooperativity is well known to promote the speed of some biochemical reactions by accelerating the activity of enzymes. Recent studies have shown that cooperative interactions also function during the formation of a complex cellular structure, the mitotic spindle. Capture of kinetochores by dynamic astral microtubules was originally proposed as the basis of spindle formation. However, mounting evidence indicates that a more complex series of events occurs. It is now clear that there are multiple microtubule nucleation and capture sites throughout the spindle. Kinetochores, centrosomes and microtubules play multiple roles in establishing connections between spindle components and integrating them into a common structure. These data support a modified search-and-capture model that incorporates additional assembly pathways coordinated by a RanGTP gradient.

Myosin at work: motor adaptations for a variety of cellular functions.

Cells have evolved multiple mechanisms to overcome the effects of entropy and diffusion to create a highly ordered environment. For cells to function properly, some components must be anchored to provide a framework or structure. Others must be rapidly transported over long distances to generate asymmetries in cell morphology and composition. To accomplish long-range transport, cells cannot rely on diffusion alone as many large organelles and macromolecular complexes are essentially immobilized by the dense meshwork of the cytosol. One strategy used by cells to overcome diffusion is to harness the free energy liberated by ATP hydrolysis through molecular motors. Myosins are a family of actin based molecular motors that have evolved a variety of ways to contribute to cellular organization through numerous modifications to the manner they convert that free energy into mechanical work.

Native Myosin-IXb is a plus-, not a minus-end-directed motor.

Myosin-IXb (Myo9b) is a single-headed, processive motor that contains a Rho-GTPase-activating protein (GAP) domain within its tail. Although tail-less myosin- IXb motor domain moves towards the minus end of the actin filament, we show here that full-length myosin-IXb is a plus-end-directed motor. This suggests that the tail domain of myosin-IXb regulates motor directionality.

Transferrin receptor recycling in the absence of perinuclear recycling endosomes.

In mammalian cells, internalized receptors such as transferrin (Tfn) receptor are presumed to pass sequentially through early endosomes (EEs) and perinuclear recycling endosomes (REs) before returning to the plasma membrane. Whether passage through RE is obligatory, however, remains unclear. Kinetic analysis of endocytosis in CHO cells suggested that the majority of internalized Tfn bypassed REs returning to the surface from EEs. To determine directly if REs are dispensable for recycling, we studied Tfn recycling in cytoplasts microsurgically created to contain peripheral EEs but to exclude perinuclear REs. The cytoplasts actively internalized and recycled Tfn. Surprisingly, they also exhibited spatially and temporally distinct endosome populations. The first appeared to correspond to EEs, labeling initially with Tfn, being positive for early endosomal antigen 1 (EEA-1) and containing only small amounts of Rab11, an RE marker. The second was EEA-1 negative and with time recruited Rab11, suggesting that cytoplasts assembled functional REs. These results suggest that although perinuclear REs are not essential components of the Tfn recycling pathway, they are dynamic structures which preexist in the peripheral cytoplasm or can be regenerated from EE- and cytosol-derived components such as Rab11.

Myosin-IXb is a single-headed and processive motor.

Class IX myosins are unique among the many classes of known actin-based motors in that the tail region of these myosins contains a GTPase-activating protein domain for the small GTP-binding protein, Rho. Previous studies on human myosin-IXb indicate that this myosin is mechanochemically active and exhibits actin-binding properties similar to the processive motor, myosin-Va. Motility analysis of antibody-tethered myosin-IXb performed using the sliding actin filament assay indicates that this myosin does exhibit properties characteristic of a processive motor. Like myosin-Va, the velocity of myosin-IXb remains constant (38.2 +/- 1.2 nm/s) even at single motor/filament densities. At low motor densities, filaments can be seen passing through and pivoting about single points on the motility surface. Analysis of filament landing rates as a function of motor density also indicates that a single motor is sufficient for filament movement. However, in contrast to myosin-Va, which uses coordinated motion of its two heads to move processively along the filament, hydrodynamic and chemical cross-linking studies indicate that under the conditions tested, myosin-IXb is a single-headed motor consisting of a single heavy chain and associated light chains.