Attachment stability and change in early childhood and associated moderators

This thesis examined attachment stability and change in parent-child relationships in the first child-specific meta-analysis and the first measurement in an Australian sample. Employing novel statistical methods, this thesis demonstrated the existence of publication bias, a low degree of attachment stability, and the importance of parental sensitivity on developmentally significant attachment transition

Lack of trust in maternal support is associated with negative interpretations of ambiguous maternal behavior

Attachment theory assumes that children who lack trust in maternal availability for support are more inclined to interpret maternal behavior in congruence with their expectation that mother will remain unavailable for support. To provide the first test of this assumption, early adolescents (9-13 years old) were asked to assess whether ambiguous interactions with mother should be interpreted in a positive or a negative way. In our sample (n = 322), results showed that early adolescents' lack of trust in their mother's availability for support was related to more negative interpretations of maternal behavior. The associations remained significant after controlling for depressive mood. The importance of these findings for our understanding of attachment theory, attachment stability, and clinical practice are discussed

CENP-F stabilizes kinetochore-microtubule attachments and limits dynein stripping of corona cargoes

Accurate chromosome segregation demands efficient capture of microtubules by kinetochores and their conversion to stable bioriented attachments that can congress and then segregate chromosomes. An early event is the shedding of the outermost fibrous corona layer of the kinetochore following microtubule attachment. Centromere protein F (CENP-F) is part of the corona, contains two microtubule-binding domains, and physically associates with dynein motor regulators. Here, we have combined CRISPR gene editing and engineered separation-of-function mutants to define how CENP-F contributes to kinetochore function. We show that the two microtubule-binding domains make distinct contributions to attachment stability and force transduction but are dispensable for chromosome congression. We further identify a specialized domain that functions to limit the dynein-mediated stripping of corona cargoes through a direct interaction with Nde1. This antagonistic activity is crucial for maintaining the required corona composition and ensuring efficient kinetochore biorientation

Mps1 regulates kinetochore-microtubule attachment stability via the ska complex to ensure error-free chromosome segregation

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1’s error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex’s conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation

Stable kinetochore-microtubule attachment is sufficient to satisfy the spindle assembly checkpoint

2016 Summer.Includes bibliographical references.During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. The kinetochore proteins that bind spindle microtubules are exquisitely regulated to ensure correct segregation of genetic material at mitotic exit. Aurora B Kinase (ABK) phosphorylates Hec1, a protein that directly binds microtubules. This is critical for enabling the release of incorrect kinetochore-microtubule attachments. Hec1 has nine ABK phosphorylation sites on its tail domain allowing for precise control over binding affinity. We find that at least 7 of these sites are required for wild-type kinetochore-microtubule (K-MT) attachment stability as evaluated by inter-kinetochore distance measures and chromosome alignment capability. We further observe that several sites may have more influence on K-MT attachment stability than others. Hec1 mutations preventing phosphorylation increase kinetochore-microtubule attachment stability. In the absence of stable kinetochore–microtubule (K-MT) attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore–microtubule attachments in human cells using a non-phosphorylatable Hec1mutant, a core component of the attachment machinery. This mutant reduced the ability of ABK to cause release of erroneous K-MT so we could test the hypothesis that stable K-MT attachments satisfy the SAC even if those attachments deviate from the canonical bipolar form. We find that stable attachments are sufficient to satisfy the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal. These results indicate a conformational change(s), within the kinetochore that occurs upon stable kinetochore-microtubule binding causes the eviction of SAC proteins. This advance in our understanding of SAC function offers insight into the mode of action and the variation in cellular response to mitotic arrest therapies often used in treatments of cancers

Optical characterization of individual liposome-loaded microbubbles

Newly developed liposome-loaded (LPS) microbubbles are characterized by comparing their oscillating response with standard phospholipid-coated (bare) microbubbles using the ultra-high speed imaging (Brandaris 128) camera. A study of the shell properties indicate nearly the same shell elasticity and a higher shell viscosity for LPS bubbles than for bare bubbles. A frequency and pressure-dependent bubble acoustical behavior study shows a higher threshold for the initiation of bubble vibrations for LPS bubbles. In addition, an “expansion-only” behavior was observed for up to 69% of the investigated LPS bubbles which mostly occurred at lower acoustic pressures (≤30 kPa). Liposome attachment stability were studied using fluorescence imaging. The internal relationship among morphological structure, shell properties and ultrasonic behavior of LPS bubbles by optical characterization facilitate preclinical study and clinical application of LPS bubbles in ultrasound triggered drug delivery system.Newly developed liposome-loaded (LPS) microbubbles are characterized by comparing their oscillating response with standard phospholipid-coated (bare) microbubbles using the ultra-high speed imaging (Brandaris 128) camera. A study of the shell properties indicate nearly the same shell elasticity and a higher shell viscosity for LPS bubbles than for bare bubbles. A frequency and pressure-dependent bubble acoustical behavior study shows a higher threshold for the initiation of bubble vibrations for LPS bubbles. In addition, an “expansion-only” behavior was observed for up to 69% of the investigated LPS bubbles which mostly occurred at lower acoustic pressures (≤30 kPa). Liposome attachment stability were studied using fluorescence imaging. The internal relationship among morphological structure, shell properties and ultrasonic behavior of LPS bubbles by optical characterization facilitate preclinical study and clinical application of LPS bubbles in ultrasound triggered drug delivery system