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Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) family of proteins, serves as a crosslinker between plasma membrane and actin cytoskeleton. It provides structural links to connect cell cortex and plasma membrane, acting as a signal transducer in multiple pathways during migration, proliferation, and development. Ezrin is also considered crucial during cancer metastasis, due to its altered expression levels and subcellular localization. Nevertheless, the mechanism how Ezrin promotes cancer progression remains unclear. In this thesis, the primary role of Ezrin phosphorylation in the relationship between cell motility, cell mechanical properties and cytoskeleton organization was investigated as follows: Firstly, long-term live-cell imaging was used to evaluate the effect of Ezrin phosphorylation on cell migration. The key results showed that cells expressing active Ezrin T567D migrated faster, with more protein accumulating at the cell rear. Secondly, the changes of cell mechanical properties caused by Ezrin were explored by atomic force microscopy (AFM). It revealed that transfection of active Ezrin T567D decreased cortical stiffness but increased cytoskeleton stiffness. Thirdly, image quantification approaches were carried out to study the effect of Ezrin on three cytoskeleton (actin filaments, microtubules and vimentin) as well as nuclear mechanical properties. The results showed that Ezrin T567D transfection led to thicker actin stress fibers and longer vimentin filaments. In the end, a sandwich model was developed to study bleb based migration in which Ezrin is also involved. Bleb based migration was observed by confining cells between two pieces of polyacrylamide gels. This model showed potential for the future investigation of Ezrin phosphorylation in bleb based migration. Altogether, we have fundamentally revealed that dynamic regulation of phosphorylated Ezrin played a pivotal role in cell motility and cell mechanical properties by altering the cytoskeleton’s microstructure. These findings are useful for a better understanding of how Ezrin is biophysically involved in cancer progression.

Every time we move our eyes, a slightly different image is recorded at the back of our eye (i.e., the retina). However, we seldom perceive this change in the visual world due to frequent eye movements or saccades. How we maintain stable perception of the world is then a critical yet complex question to understand our visual system and cognitive processes. My dissertation aims at understanding more about visual stability from three example visual functions that interact with saccades. Chapter 1 (General Introduction) briefly summarizes the relevant literature on visual stability. Chapter 2 explores the brain representations of top-down spatial attention in retinotopic (eye-centered) and spatiotopic (world-centered) reference frames across saccades. By analyzing the patterns of brain activity from fMRI scans, I found that updating spatial attention across saccades might be coded with a combination of attention “shift” and “hold” signals across different brain regions/networks. Chapter 3 focuses on how saccades affect the perception and neural representations of 3D stimulus depth. Participants passively viewed stimuli at different 3D locations, and the brain representations of vertical and depth information were assessed and compared between no-saccade blocks and saccade blocks. The results showed that in saccade blocks, despite the retinal changes in horizontal position induced by the saccades, both vertical and depth information could still be decoded in visual areas, to a similar extent as in no-saccade blocks. In contrast, little vertical or depth information could be decoded across no-saccade blocks with different eye positions in any visual areas, indicating eye-position-dependency during stable fixation. Representations of spatial locations (2D and depth) may become more tolerant of eye positions during “dynamic” saccades, perhaps due to active remapping during saccades which may encourage more stable representations of the world. Chapter 4 presents a human behavioral study focused on target localization, where participants remembered and reported the location of the target on the screen after making a saccade, with task-irrelevant nontarget objects presented alongside. The results showed that the spatial relationship between the target and nontargets was utilized for target localization; however it also biased responses spatially. The final Chapter 5 (General Discussion) discusses how these three studies together help to further understand how visual information is integrated despite changeable inputs due to eye movements.

While we are frequently moving our eyes, it is important to keep our visual perception stable. To maintain visual stability, we use information from both retinal and extra-retinal sources. Retinal information can include “targets” – objects on which people fixate their eyes or perform cognitive tasks – which are directly related with the current cognitive goal and consume a large part of the resources. Other objects that are currently not the fixation or the task-related target, i.e., “nontargets”, can act as “landmarks” to influence target localization. However, it has been less systematically examined how people actually use these nontargets to facilitate performance after moving their eyes. This project investigated whether the presence of nontargets could facilitate target localizing performance, and if so, how participants would use the nontarget information. The results showed that participants used nontarget information in a target location recall task but not a target displacement detection task. In the recall task, nontargets appeared to be used in two ways: by providing relational information in space to reduce localization errors, and also by helping to counteract localization biases related to the saccade. We argue that nontargets can facilitate target localization after saccades when needed, possibly by serving as anchors for the target.

Patients with Rett Syndrome (RTT) show severe breathing disorders in addition to other neuropathological features, contributing to the high incidence of sudden unexplained death and abnormal brain development. However, the molecular and cellular mechanisms underlying the breathing disorders are still unknown. Recent studies indicate that the dysfunction of brainstem norepinephrine (NE) systems are closely associated with breathing disorders in RTT patients as well as its mice model, the Mecp2-null (Mecp2─/Y) mice. This as well as the fact the major group of NE-ergic neurons in the locus coeruleus (LC) is CO2 chemosensitive suggests that the breathing disorders in RTT may be related these LC neurons. To test this hypothesis, we took a multidisciplinary approach and systematically studied these neurons using molecular biology, in-vitro brain slices, acutely dissociated neurons, immunocytochemistry, and whole-body plethysmograph. To facilitate the electrophysiological studies, we developed a new strain of transgenic mice with GFP expression selectively in the LC neurons of both WT and Mecp2─/Y mice. Breathing activity of the Mecp2─/Y mice showed selective disruptions in responses to mild hypercapnia. The defect was alleviated with the NE uptake blocker desipramine, suggesting the involvement of NE in central CO2 chemosensitivity. In the LC region, the expressions of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) at both protein and mRNA levels reduced by ~50% in Mecp2─/Y mice. No evidence was found for selective deficiency in TH- or DBH-containing neurons in Mecp2─/Y mice, and no major loss of NE-ergic LC cells were found, indicating that the NE defect is likely to result from deficient expression of biosynthetic enzymes rather than a loss of neurons in the LC. Several intrinsic membrane properties were abnormal in Mecp2─/Y LC neurons in comparison to wild type cells, including stronger inward rectification, shorter time constant, extended action potential duration, smaller amplitude of medium afterhyperpolarization (AHP) and over-expression of fast AHP. These abnormalities seem to be associated with the altered K+ and Na+ currents. Most importantly, Mecp2─/Y LC neurons displayed defective CO2 chemosensitivity in agreement of in vivo CO2 response, likely due to excessive expression of the homomeric Kir4.1 channel. Thus, it seems that the global effect of MeCP2 on the A6 NE system contributes to the impaired systemic CO2 response as well as the breathing irregularities in Mecp2─/Y mice. Such an alteration allowed CO2 to be detected only when hypercapnia became severe, leading to periodical hyper- and hypoventilation. These findings not only provide a novel etiology for the breathing disturbances of Mecp2─/Y mice but also show direct evidence for the first time on a molecular mechanism for the central CO2 chemosensitivity.