Roles of Lissencephaly Gene, LIS1, in Regulating Cytoplasmic Dynein Functions: a Dissertation

Spontaneous mutations in the human LIS1 gene are responsible for Type I lissencephaly ( smooth brain ). The distribution of neurons within the cerebral cortex of lissencephalic children appears randomized, probably owing to a defect in neuronal migration during early development. LIS1 has been implicated in the dynein pathway by genetic analyses in fungi. We previously reported that the vertebrate LIS1 co-localized with dynein at prometaphase kinetochores, and interference with LIS1 function at kinetochore caused misalignment of chromosomes onto the metaphase plate. This leads to a hypothesis that LIS1 might regulate kinetochore protein targeting. In order to test this hypothesis, I created dominant inhibitory constructs of LIS1. After removal of the endogenous LIS1 from the kinetochore by overexpression of the N-terminal self-association domain of LIS1, dynein and dynactin remained at the kinetochores. This result indicated that LIS1 is not required for dynein to localize at the kinetochore. Next, CLIP-170 was displaced from the kinetochores in the LIS1 full-length and the C-terminal WD-repeat overexpressers, suggesting a role for LIS1 in targeting CLIP-170 onto kinetochores. LIS1 was co-immunoprecipitated with dynein and dynactin. Its association with kinetochores was mediated by dynein and dynactin, suggesting LIS1 might interact directly with subunits of dynein and/or dynactin complexes. I found that LIS1 interacted with the heavy and intermediate chains (HC and IC) of dynein complex, and the dynamitin subunit of dynactin complex. In addition to kinetochore targeting, the LIS1 C-terminal WD-repeat domain was responsible for interactions with dynein and dynactin. Interestingly, LIS 1 interacted with two distinct sites on HC: one in the stem region containing the subunit-binding domain, and the other in the first AAA motif of the motor domain, which is indispensable for the ATPase function of the motor protein. This LIS1-dynein motor domain interaction suggests a role for LIS1 in regulating dynein motor activity. To test this hypothesis, changes of dynein ATPase activity was measured in the presence of LIS1 protein. The ATPase activity of dynein was stimulated by the addition of a recombinant LIS1 protein. Besides kinetochores, others and we have found LIS1 also localized at microtubule plus ends. LIS1 may mediate dynein and dynactin mitotic functions at these ends by interacting with astral microtubules at cortex, and associating with the spindle microtubules at kinetochores. Overexpression of LIS1 displaced dynein and dynactin from the microtubule plus ends, and mitotic progression was severely perturbed in LIS1 overexpressers. These results suggested that the role for LIS1 at microtubule plus ends is to regulate dynein and dynactin interactions with various subcellular structures. Results from my thesis research clearly favored the conclusion that LIS1 activates dynein ATPase activity through its interaction with the motor domain, and this activation is important to establish an interaction between dynein and microtubule plus ends during mitosis. I believe that my thesis work not only has provided ample implications regarding dynein dysfunction in disease formation, but also has laid a significant groundwork for more future studies in regulations of the increasing array of dynein functions

Experimental Study on Hydraulic Jumps with and Without Sediment

Source: ICHE Conference Archive - https://mdi-de.baw.de/icheArchive

Description of local dilatancy and local rotation of granular assemblies by microstretch modeling

AbstractThis study investigates the microstretch continuum modeling of granular assemblies while accounting for both the dilatant and rotational degrees of freedom of a macroelement. By introducing the solid volume fraction and the gyration radius of a granular system, the balance equations of the microstretch continuum are transformed into a new formulation of evolution equations comprising six variables: the solid volume fraction, the gyration radius, the velocity field, the averaged angular velocity, the rate of gyration radius, and the internal energy. The bulk microinertia density, the averaged angular velocity, and the microgyration tensor at a macroscopic point are obtained in terms of discrete physical quantities. The bulk part and the rotational part of the microgyration tensor are proposed as the two indices to measure the local dilatancy and local rotation of granular assemblies. It is demonstrated in the numerical simulation that the two indices can be used to identify the shear band evolution in a granular system under a biaxial compression

N-Cadherin, Spine Dynamics, and Synaptic Function

Dendritic spines are one-half (the postsynaptic half) of most excitatory synapses. Ever since the direct observation over a decade ago that spines can continually change size and shape, spine dynamics has been of great research interest, especially as a mechanism for structural synaptic plasticity. In concert with this ongoing spine dynamics, the stability of the synapse is also needed to allow continued, reliable synaptic communication. Various cell-adhesion molecules help to structurally stabilize a synapse and its proteins. Here, we review the effects of disrupting N-cadherin, a prominent trans-synaptic adhesion molecule, on spine dynamics, as reported in Mysore et al. (2007). We highlight the novel method adopted therein to reliably detect even subtle changes in fast and slow spine dynamics. We summarize the structural, functional, and molecular consequences of acute N-cadherin disruption, and tie them in, in a working model, with longer-term effects on spines and synapses reported in the literature

Effects of N-Cadherin Disruption on Spine Morphological Dynamics

Structural changes at synapses are thought to be a key mechanism for the encoding of memories in the brain. Recent studies have shown that changes in the dynamic behavior of dendritic spines accompany bidirectional changes in synaptic plasticity, and that the disruption of structural constraints at synapses may play a mechanistic role in spine plasticity. While the prolonged disruption of N-cadherin, a key synaptic adhesion molecule, has been shown to alter spine morphology, little is known about the short-term regulation of spine morphological dynamics by N-cadherin. With time-lapse, confocal imaging in cultured hippocampal neurons, we examined the progression of structural changes in spines following an acute treatment with AHAVD, a peptide known to interfere with the function of N-cadherin. We characterized fast and slow timescale spine dynamics (minutes and hours, respectively) in the same population of spines. We show that N-cadherin disruption leads to enhanced spine motility and reduced length, followed by spine loss. The structural effects are accompanied by a loss of functional connectivity. Further, we demonstrate that early structural changes induced by AHAVD treatment, namely enhanced motility and reduced length, are indicators for later spine fate, i.e., spines with the former changes are more likely to be subsequently lost. Our results thus reveal the short-term regulation of synaptic structure by N-cadherin and suggest that some forms of morphological dynamics may be potential readouts for subsequent, stimulus-induced rewiring in neuronal networks

Activity-Regulated N-Cadherin Endocytosis

SummaryEnduring forms of synaptic plasticity are thought to require ongoing regulation of adhesion molecules, such as N-cadherin, at synaptic junctions. Little is known about the activity-regulated trafficking of adhesion molecules. Here we demonstrate that surface N-cadherin undergoes a surprisingly high basal rate of internalization. Upon activation of NMDA receptors (NMDAR), the rate of N-cadherin endocytosis is significantly reduced, resulting in an accumulation of N-cadherin in the plasma membrane. β-catenin, an N-cadherin binding partner, is a primary regulator of N-cadherin endocytosis. Following NMDAR stimulation, β-catenin accumulates in spines and exhibits increased binding to N-cadherin. Overexpression of a mutant form of β-catenin, Y654F, prevents the NMDAR-dependent regulation of N-cadherin internalization, resulting in stabilization of surface N-cadherin molecules. Furthermore, the stabilization of surface N-cadherin blocks NMDAR-dependent synaptic plasticity. These results indicate that NMDAR activity regulates N-cadherin endocytosis, providing a mechanistic link between structural plasticity and persistent changes in synaptic efficacy

Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function

Mutations in the human LIS1 gene cause type I lissencephaly, a severe brain developmental disease involving gross disorganization of cortical neurons. In lower eukaryotes, LIS1 participates in cytoplasmic dynein-mediated nuclear migration. We previously reported that mammalian LIS1 functions in cell division and coimmunoprecipitates with cytoplasmic dynein and dynactin. We also localized LIS1 to the cell cortex and kinetochores of mitotic cells, known sites of dynein action. We now find that the COOH-terminal WD repeat region of LIS1 is sufficient for kinetochore targeting. Overexpression of this domain or full-length LIS1 displaces CLIP-170 from this site without affecting dynein and other kinetochore markers. The NH2-terminal self-association domain of LIS1 displaces endogenous LIS1 from the kinetochore, with no effect on CLIP-170, dynein, and dynactin. Displacement of the latter proteins by dynamitin overexpression, however, removes LIS1, suggesting that LIS1 binds to the kinetochore through the motor protein complexes and may interact with them directly. We find that of 12 distinct dynein and dynactin subunits, the dynein heavy and intermediate chains, as well as dynamitin, interact with the WD repeat region of LIS1 in coexpression/coimmunoprecipitation and two-hybrid assays. Within the heavy chain, interactions are with the first AAA repeat, a site strongly implicated in motor function, and the NH2-terminal cargo-binding region. Together, our data suggest a novel role for LIS1 in mediating CLIP-170–dynein interactions and in coordinating dynein cargo-binding and motor activities

Polyploids require Bik1 for kinetochore–microtubule attachment

The attachment of kinetochores to spindle microtubules (MTs) is essential for maintaining constant ploidy in eukaryotic cells. Here, biochemical and imaging data is presented demonstrating that the budding yeast CLIP-170 orthologue Bik1is a component of the kinetochore-MT binding interface. Strikingly, Bik1 is not required for viability in haploid cells, but becomes essential in polyploids. The ploidy-specific requirement for BIK1 enabled us to characterize BIK1 without eliminating nonhomologous genes, providing a new approach to circumventing the overlapping function that is a common feature of the cytoskeleton. In polyploid cells, Bik1 is required before anaphase to maintain kinetochore separation and therefore contributes to the force that opposes the elastic recoil of attached sister chromatids. The role of Bik1 in kinetochore separation appears to be independent of the role of Bik1 in regulating MT dynamics. The finding that a protein involved in kinetochore–MT attachment is required for the viability of polyploids has potential implications for cancer therapeutics

Discovery of New Eunicellins from an Indonesian Octocoral Cladiella sp.

Two new 11-hydroxyeunicellin diterpenoids, cladieunicellin F (1) and (–)-solenopodin C (2), were isolated from an Indonesian octocoral Cladiella sp. The structures of eunicellins 1 and 2 were established by spectroscopic methods, and eunicellin 2 was found to be an enantiomer of the known eunicellin solenopodin C (3). Eunicellin 2 displayed inhibitory effects on the generation of superoxide anion and the release of elastase by human neutrophils. The previously reported structures of two eunicellin-based compounds, cladielloides A and B, are corrected in this study