<p>Dendrite shape is a defining component of neuronal function. Yet, the mechanisms specifying diverse dendritic morphologies, and the extent to which their functioning depends on these morphologies, remain unclear. Here, we demonstrate a dendrite-specific requirement for the microtubule severing protein Katanin p60-like 1 (Kat-60L1) in regulating the elaborate branch morphology and nocifensive functions of Drosophila melanogaster larval class IV dendritic arborization (da) neuron dendrites. Through genetic loss of function analysis we show that loss of kat-60L1 reduced dendrite branching and process length, particularly during a period of normally extensive growth. This morphological defect was paralleled by a reduction in nocifensive responsiveness mediated by these neurons, indicating a tight correlation between neuronal function and the full extent of the dendritic arbor. To understand the mechanism underlying Kat-60L1's effects, we used in vivo imaging of the microtubule plus-end binding protein EB1, and found fewer polymerizing microtubules within mutant dendrites. Kat-60L1 thus promotes microtubule growth within class IV dendrites to establish the full arbor complexity and nocifensive functions of these neurons. </p><p>Although reduction of the related microtubule severing protein Spastin also compromised class IV dendrite arborization and nocifensive responses, microtubule polymerization in dendrites was unchanged in spastin mutants, and behavioral defects arose from generally compromised neuronal excitation. Kat-60L1 and Spastin thus function in distinct neuronal compartments to establish the complex dendritic morphology and sensory functions of class IV da neurons via distinct mechanisms of microtubule regulation. Whereas Spastin regulates stable microtubules affecting both pre- and post-synaptic compartments of these neurons, Kat-60L1 function is required specifically in dendrites to promote their complex arborization through the addition of growing microtubule numbers. Double mutant analysis demonstrated that Kat-60L1 and Spastin function antagonistically to promote dendritic aborization, likely involving other molecular players involved in regulating the microtubule cytoskeleton. Lastly, we identified Mi-2 as a transcriptional regulator of both kat-60L1 and spastin and show a genetic interaction between mi-2 and kat-60L1 in the class IV dendritic arbor, demonstrating that Mi-2 antagonizes Kat-60L1 function, possibly through the parallel upregulation of spastin. These data support a key role for the differential utilization of microtubule severing in generating distinct neuronal morphologies and subsequent function.</p> / Dissertation

Glioblastomas are the most common and the deadliest types of brain tumours. Due to their highly invasive behaviour, they are incurable by convencial therapeutical strategies. It was shown that some components of microtubules, namely class III β-tubulin, γ-tubulin and microtubule severing protein spastin are overexpressed in glioblastoma cell lines as well as glioblastomas. This diploma thesis is focused on the expression, subcellular distribution and function of katanin, another microtubule-severing enzyme, in gliobastoma cell lines. Katanin is formed by catalytic (p60) and regulatory (p80) subunits. Expression and cellular localization of both katanin subunits was studied in panel of human glioblastoma cell lines isolated form adults (T98G, U87MG, U118MG and U138 MG) and child (KNS42). Data presented in this thesis demonstrated that katanin subunits were overexpresed both on transcript and protein levels in T98G, U87MG and KNS42 cell lines, but not in U138MG and U118MG cell lines when compared to normal non- transformed human astrocytes. Immunofluorescence microscopy revealed that both katanin subunits were diffusively distributed in cytoplasm and concentrated on spindle poles of mitotic cells and on leading edges of migrating cells. Examination of cell motility revealed that velocities in...

No description available.

The cytoskeleton is a dynamic network of microtubules constantly being reorganized to meet the spatiotemporal demands of the cell. Microtubules are organized into subcellular highways to control cell processes such as cell division, cargo transport, and neuronal development and maintenance. Reorganization of this intricate network is tightly regulated by various stabilizing and destabilizing microtubule-associated proteins that decorate the network. Katanin p60 is a microtubule destabilizing enzyme from the ATPases Associated with various Activities (AAA+) family. It can both sever and depolymerize microtubules. In order to sever microtubules, katanin recognizes the tubulin carboxy-terminal tails (CTTs) and hydrolyzes ATP. Using super-resolution microscopy and image analysis, we find that the tubulin CTTs are not required for katanin to depolymerize microtubules. We also characterize the regulation of microtubule severing and depolymerization by katanin in various nucleotide states. A better understanding of how CTTs and nucleotides regulate microtubule severing and depolymerization by katanin will help future research aimed to correct katanin activity when these processes goes awry as in improper chromosome segregation during mitosis or loss of microtubule integrity in neuronal diseases.

Microtubules (MT) are regulated by multiple categories of proteins, including proteins responsible for severing MTs that are therefore called MT-severing proteins. Studies of katanin, spastin, and fidgetin in animal systems have clarified that these proteins are MT-severing. However, studies in plants have been limited to katanin p60, and little is known about spastin or fidgetin and their function in plants. I looked at plant genomes to identify MT-severing protein homologues to clarify which severing proteins exist in plants. I obtained data from a variety of eukaryotic species to look for MT-severing proteins using homology to human proteins and analyzed these protein sequences to obtain information on the evolution of MT-severing proteins in different species. I focused this analysis on MT-severing proteins in the maize and Arabidopsis thaliana genomes. I created evolutionary phylogenetic trees for katanin-p60, katanin-p80, spastin, and fidgetin using sequences from animal, plant, and fungal genomes. I focused on Arabidopsis spastin and worked to understand its functionality by identifying protein interaction partners. The yeast two-hybrid technique was used to screen an Arabidopsis cDNA library to identify putative spastin interactors. I sought to confirm the putative protein interactions by using molecular tools for protein localization such as the YFP system. Finally, a Biomolecular Fluorescence Complementation (BiFC) assay was initiated as a proof of concept for confirmation of in vivo protein-protein interaction.

Understanding how the cellular cytoskeleton is maintained and regulated is important to elucidate the functions of many structures such as the mitotic spindle, cilia and flagella. Katanin p60, microtubule-severing enzymes from the ATPase associated with cellular activities (AAA+) family, has previously been shown in our lab to be inhibited by free tubulin as well as α- and β-tubulin carboxy-terminal tail (CTT) constructs. Here we investigate the inhibition ability of several different tubulin CTT sequences. We quantify the effect of the addition of these constructs on the severing and binding activity of katanin. We find that some constructs inhibit katanin better than others and two constructs that appear to enhance katanin activity. Our findings add nuance to our previous findings that consensus α-tubulin tails are less inhibitory of katanin than consensus β-tubulin [3]. Surprisingly, we find that a polyglutamate sequence activates katanin while it has previously been shown to inhibit spastin, a different microtubule-severing enzyme associated with the neuromuscular disease Hereditary Spastic Paraplegia [23]. These results highlight that different CTT sequences can control the activity of severing enzymes and ultimately affect the cytoskeletal network organization in a cell type and location-dependent manner.

No description available.

No description available.