Peroxisomes are ubiquitous organelles found in almost all eukaryotes. They are sensitive to changes in cellular homeostasis and involved in various metabolic processes. Deficiencies in peroxisome function cause severe neurological problems. Here I report, investigation of peroxisome motility and its relation to peroxisomal functions in the fungal model system Ustilago maydis. Peroxisomes are mostly motile in Ustilago maydis. Motile peroxisomes show different motility patterns: short-range pulse type movements and long range bidirectional motility. Motility behaviour is not static as oscillating peroxisomes may start long-range motility. Here, I present evidence that long-range bidirectional peroxisome motility is an energy driven process and is essential for homogeneous distribution of peroxisomes. Similar to early endosomes and endoplasmic reticulum, microtubule motors kinesin-3 and dynein are responsible for long-range peroxisome transport. In addition to using the same molecular motors for transport, early endosomes, endoplasmic reticulum and peroxisomes have the same transport velocity. Interestingly, motile peroxisomes and endoplasmic reticulum tubules co-localize with early endosomes. Functional investigation of early endosome mutants, Δrab5a and Yup1ts has revealed a novel transport mechanism where endoplasmic reticulum and peroxisomes hitch hike on early endosomes. Additionally, I report functional characterization of an AAA-ATPase, um05592, which has high homology to human protein NP_055873. Altogether these results reveal molecular mechanism of peroxisome transport in Ustilago maydis. Similarities in transport machinery illustrate Ustilago maydis as a model system to study peroxisome function in mammalian cells.

No description available.

Proper attachment of chromosomes to microtubules is important for the accurate segregation of chromosomes and genome stability. The initial engagement of chromosomes happens along the lateral wall of microtubules through a highly specialised protein structure assembled on the centromeric DNA, the kinetochore. Ultimately, kinetochores must be attached to the ends of microtubules (a geometry called end- on attachment). A series of highly dynamic steps called the end-on conversion process, converts the initial immature lateral attachments into mature end-on attachments. How this process is finely tuned by phosphorylation and dephosphorylation to achieve stable attachments is still unclear. Furthermore, what is the role of microtubule-associated proteins in the stabilisation of kinetochore-microtubule attachments is unknown. This project aimed to study the role of phosphatases in the regulation of the end-on conversion process. First, I investigated the different contribution of the two outer-kinetochore phosphatases - BubR1- recruited PP2A-B56 and KNL1-recruited PP1 - in counteracting Aurora B kinase during the end-on conversion process. I found that BubR1-recruited PP2A-B56 plays an essential role in the process, but KNL1-recruited PP1 does not. I also investigated whether the HEC1/Ndc80 N-tail is a critical substrate of Aurora B phosphorylation for the stabilisation of the end-on attachments. Using a phospho-dead mutant of the HEC1/Ndc80 N-tail, I discovered that cells are still susceptible to Aurora B activity, indicating downstream pathways independent of HEC1/Ndc80. Then, I studied the biological role of the Astrin C-terminus, where an evolutionarily conserved RVMF motif, a putative PP1 binding site, is located. My findings show C-terminal Astrin mutants fail to localise at kinetochores of both monopolar and bipolar spindles; induce defects in the end-on conversion process in monopolar spindles and prolong mitosis time with increased Mad2 levels at the outer-kinetochore. A kinase inhibitor assay showed that kinetochore-microtubule attachment defects in Astrin mutant expressing cells could be rescued when both Aurora B and Cdk1 kinases are inhibited, suggesting a role for Astrin’s C-terminus in counteracting Aurora B and Cdk1 activity. Finally, I probed the putative interaction of the Astrin C-terminus and PP1 using biochemistry, cell biology and fluorescence microscopy techniques. I discovered that artificially targeting PP1 onto the Astrin C-terminus but not on the N-terminus rescues mutants localisation defects at the kinetochore. In summary, my results indicate that Astrin and PP1 interact at the kinetochore of living cells. In conclusion, my work shows that mitotic phosphatases have distinctive contributions in the regulation of the dynamic steps of the end-on conversion process and that Astrin is a potential PP1 phosphatase recruiter at the outer-kinetochore, where is necessary for the stabilisation of end-on attachments.

Mto1 recruits γ-tubulin to the sites of cytoplasmic microtubule nucleation in the fission yeast Schizosaccharomyces pombe. The regulation of Mto1 localisation is central to re-modelling of the microtubule cytoskeleton during the cell cycle. This thesis describes how Mto1 is recruited to the cell equator during mitosis, thereby establishing the equatorial microtubule nucleation centre (eMTOC). F-actin is found to be required for Mto1 localisation to the cell equator and Mto1 is shown to co-localise with the cytokinetic actin ring (CAR). Yeast 2-hybrid screening and tandem-affinity purification were used to screen for additional proteins required for Mto1 localisation to the equator. Further candidate screening identified Myp2, a type II myosin present in the CAR, as being required for Mto1 localisation to the cell equator. Myp2 is shown to physically interact with Mto1 and to be required for formation of the post-anaphase microtubule array. The regulation of Mto1 localisation to the cell equator was also studied. Time-lapse microscopy reveals that Mto1 localisation to the equator does not require either the anaphase-promoting complex or the septation initiation network, both of which have been previously shown to be necessary for the recruitment of γ-tubulin to the eMTOC. Maintenance of the equatorial CAR has previously been attributed to the postanaphase array. The position of the CAR in the mto1-427 mutant strain, which fails to nucleate a PAA, is shown to be unaltered from wild-type strain during exponential growth, suggesting that the PAA does not centre the CAR during normal growth.

The microtubule [MT] cytoskeleton of S. pombe is a highly dynamic network of filaments that facilitates intracellular transport, determines cell polarity and plays an essential role in chromosome separation during mitosis. In fission yeast, MTs are nucleated in a temporally and spatially regulated manner from sites called Microtubule Organising Centres [MTOCs], through the activity of both the g-tubulin complex [g-TuC] and the Mto1/2 complex. The Mto1/2 complex determines the localisation of the g-TuC at MTOCs, which change throughout the cell cycle. As cells enter mitosis the cytoplasmic array of MT bundles depolymerise. They are replaced by the intranuclear mitotic spindle and cytoplasmic spindle pole bodyderived astral MTs that in turn give way to the formation of the post-anaphase array. Although much is known about the properties of each type of MT array, the mechanism by which the timing of MT nucleation at different MTOCs is regulated over the cell cycle remains unclear. In the Mto1/2 complex, Mto1 is thought to provide the primary interaction with the g-TuC, and Mto2 functions by reinforcing this interaction. Due to the lack of structural information for the Mto1/2 complex, the molecular mechanism of Mto1/2- mediated assembly of the g-TuC at MTOCs is unknown. The aim of my study is to investigate the possibility that the Mto1/2 complex is able to promote g-TuC assembly by forming a direct template. In addition, I will attempt to determine the molecular role of Mto2 within the Mto1/2 complex and examine ways in which regulation of Mto2 may influence the function the Mto1/2 complex at specific MTOCs. As part of the investigation into the mechanism of Mto2 function, an in vitro analysis of recombinant protein demonstrated that in the absence of Mto1, purified Mto2 is able to self-interact as a tetramer. I have confirmed this interaction in vivo and have also shown that Mto2 forms a dimer as cells enter mitosis. However, in the context of an Mto1/2 complex the significance of the change in Mto2 oligomeric state remains unknown. Hydrodynamic analysis of a truncated form of the Mto1/2 complex suggests that it may form a heterotetramer, a hypothesis which is consistent with the equimolar levels of Mto2 and Mto1 protein within the cell. This information provides some structural insight as to how the Mto1/2 complex may interact with the g-TuC at MTOCs. Further analysis of the Mto1/2 complex revealed that in vivo, the Mto1-Mto2 interaction is disrupted during mitosis. This was found to correlate with the hyperphosphorylation of Mto2, which occurs as cells enter mitosis. Subsequently, an in vitro kinase assay demonstrated that phosphorylation of the Mto1/2 complex reduces the stability of the complex. Mass spectrometry techniques and sequence conservation were used to identify several phosphorylated residues within Mto2 and the ability of these mutants to bind to Mto1 was analysed in vivo and in vitro. In summary, in this study I have uncovered a mechanism which allows fission yeast cells to regulate the nucleation of cytoplasmic MT nucleation in a cell-cycle dependent manner, through a phosphorylation-dependent remodelling of the Mto1/2 complex.

In fission yeast, microtubule (MT) nucleation is regulated in space and time. In interphase, MTs are nucleated in the cytoplasm to regulate cell polarity, whereas in mitosis, nucleation takes place inside the nucleus to form a mitotic spindle. We hypothesize that several non-exclusive mechanisms may be responsible for this differential regulation of MT nucleation. Two fission yeast proteins, Mto1 and Pcp1, are involved in MT nucleation in interphase and mitosis, respectively. These proteins share a sequence motif, called CM1 that is responsible for interaction with the γ-tubulin complex (γ-TuC). In the first part of my project, I tested whether sequence differences between Mto1 and Pcp1 CM1 regions contribute to the differential regulation of MT nucleation in interphase vs. mitosis. I showed that the two CM1 regions are interchangeable and play no role in differential regulation of Mto1 and Pcp1. By generating Pcp1-9A1 mutant, where conserved residues within the Pcp1 CM1 region was replaced with alanines, I showed that Pcp1 CM1 region is required for its function. Moreover, using CM1 regions from two human proteins that are implicated in schizophrenia and microcephaly development, MMGL and CDK5RAP2, I showed that human CM1 domains could rescue yeast protein function, demonstrating that the CM1 region is conserved across evolution. In the second part of my project, I focused on regulation of cytoplasmic MT nucleation. In fission yeast, cytoplasmic MT nucleation occurs from several distinct sites in the cell and is promoted by the Mto1/2 complex. The Mto1/2 complex is composed of multiple copies of Mto1 and Mto2 and interacts with the γ-TuC. Disruption of the interaction of Mto1/2 with the γ-TuC, or of the Mto1-Mto2 interaction, results in a complete loss of interphase cytoplasmic nucleation. As cells enter mitosis, Mto2 is hyperphosphorylated, and the Mto1-Mto2 interaction is disrupted, leading to abolishment of cytoplasmic nucleation. This led to a hypothesis that Mto2 phosphorylation regulated the Mto1/2 complex mitotic disassembly. I showed that Mto2 phosphorylation is used to control levels of cytoplasmic nucleation in both interphase and mitosis. During interphase, I found that Mto2 is phosphorylated in order to reduce levels of MT nucleation. When Mto2 phosphorylation is prevented by mutation of phosphorylatable residues to alanines, Mto1/2 mutant complexes show a more robust interaction with the γ-TuC, and more MTs are nucleated in the cytoplasm. During mitosis, hyperphosphorylation of Mto2 plays a role in the disassembly of Mto1/2 complexes. In particular, while the interaction of wild-type Mto2 with Mto1 is disrupted during mitosis, Mto2-alanine mutants, in which phosphorylation was nearly abolished, are still able to interact with Mto1 in mitosis. Interestingly, Mto1/2 complexes containing Mto2-alanine mutants are still disassembled in mitosis by disruption of Mto2 self-interaction. I used SILAC phosphoproteomics to show that Mto2-alanine is still phosphorylated in mitosis, suggesting the Mto2 self-interaction might also be controlled by phosphorylation. While doing so, I developed a novel SILAC quantification method that is particularly useful for quantification of multiply phosphorylated proteins and peptides. Using data obtained by SILAC, I generated additional Mto2 alanine mutants with more phosphorylation sites mutated. Preliminary analysis showed that these mutants are similar to the alanine mutants analysed previously; however, more analysis is required to generate more definitive conclusions. In summary, in this study I have uncovered the functional conservation of the CM1 region from yeast to human. I also showed that Mto2 phosphorylation regulates cytoplasmic MT nucleation in both interphase and mitosis, by regulating the Mto2-Mto1 interaction and the Mto2-Mto2 self-interaction and therefore remodelling the Mto1/2 complex.

The thesis is divided in three parts based largely on published articles or on manuscripts submitted for publication. First we propose a new method which is called the shooting-bead method. This method is a fast and easy experimental technique for evaluating cantilever stiffness and flexural rigidity of semi-flexible to semi-rigid rod-like biological and nano-filaments based on the measurement of just two distances. The method is based on applying a force normal to the filament with a microsphere bead trapped in the laser tweezer followed by its sudden release. Through a simple measurement of the distances that the bead moves, the flexural rigidity of the filament can be found from the formula derived in this paper. Then we take into account the effects of the viscous drag force exerted on the filament itself. To this end, we have defined a key variable, called the filament energy-loss factor (or filament drag factor) that accounts for all the energy-loss effects. It has been shown that the effect due to the consideration of filament energy-loss factor on calculation of the flexural rigidity increases with increasing the flexibility of the filament. Finally, in the third part we discuss the effect of ultrasound on the microtubules. Here we have analytically solved equations of motion for the vibrational dynamics of an MT that is attached at its two ends. This is especially relevant for MTs during mitosis when they attach to chromosomes and centrosomes. Our analysis applies to MTs present inside a viscous solution and when driven by an ultrasound plane wave. We have shown that with using ultrasound plane waves the resonance condition for the MT treated as a rigid rod cannot be provided, and in order to achieve resonance we should excite a single mode of the MT with a harmonic number larger than a threshold value introduced in this thesis. Single mode excitation not only helps to transfer the minimum amount of energy to the surrounding medium compared with multi-mode excitation but it also allows for a simultaneous high-amplitude and high-quality factor which is impossible when using plane waves.

My initial aim was a functional analysis of the conserved Op18/stathmin family of microtubule-regulators, which includes the ubiquitous cytosolic Op18 protein and the neural membrane-attached RB3 and SCG10 proteins. The solved X-ray structure has shown that these proteins form a complex with tubulin -heterodimers via two imperfect helical repeats, which result in two head-to-tail aligned heterodimers in a tandem-tubulin complex. We have analyzed GTP exchange and GTP hydrolysis at the two exchangeable GTP-binding sites (E-site) within the tandem-tubulin complex. A comparison of Op18, RB3 and SCG10 proteins indicates that Op18/Stathmin family proteins have evolved to maintain the two heterodimers in a configuration that restrains the otherwise potent GTPase productive interactions facilitated by the head-to-head alignment of heterodimers in protofilaments. We concluded from these studies that tubulin heterodimers in complex with Op18/stathmin family members are subject to allosteric effects that prevent futile cycles of GTP hydrolysis. To understand the significance of the large differences in tubulin affinity of Op18, RB3 and SCG10, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell-surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. We showed that, in contrast to CD2-Op18, both the CD2-SCG10 and CD2-RB3 chimeras sequester tubulin at the plasma membrane, which results in >35% reduction of cytosolic tubulin heterodimer levels. However, all three CD2-chimeras, including the tubulin sequestration-incompetent CD2-Op18, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during the interphase, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules. Sm16/SmSLP (Stathmin-Like Protein) has been identified as a protein released during skin penetration of the Schistosoma mansoni parasite. This protein has been ascribed both anti-inflammatory activities and a functional similarity with the conserved cytosolic tubulin-binding protein stathmin/Op18. However, our studies refuted any functional similarity with stathmin/Op18 and we found instead that Sm16/SmSLP is a lipid bilayer binding protein that is taken up by cells through endocytosis. To study immuno-modulatory properties of Sm16/SmSLP, we designed an engineered version with decreased aggregation propensity, thus facilitating expression and purification of a soluble Sm16 /SmSLP protein from the eukaryotic organism Pichia pastoris. Determination of the hydrodynamic parameters revealed that both the recombinant and native Sm16/SmSLP is a ~9-subunits oligomer. The recombinant protein was found to have no effect on T lymphocyte activation, cell proliferation or the basal level of cytokine production of whole human blood or monocytic cells. Interestingly, however, recombinant Sm16 was found to potently inhibit the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and Poly(I:C). Since Sm16 specifically inhibits degradation of the IRAK1 signaling protein in LPS stimulated monocytes, it seems likely that inhibition is exerted proximal to the TLR-complex.

Cell walls are a distinct feature of plants and their chemical constituents, cellulose, hemicelluloses and lignin, are economically valuable. Plant fibres rich in cellulose, which mainly resides in their cell wall, are traditionally used in making paper and textiles. The changing global economic situation and environmental concerns have imparted necessity for renewable, but at the same time value added cellulosic materials. The Department of Wood Biotechnology, KTH together with its collaborators, have established EST libraries and performed transcript profiling during wood development in poplar, a tree considered as a model for wood development. The majority of the genes upregulated during cellulose biosynthesis encode proteins with known or predictable functions, such as carbohydrate active enzymes (CAzymes). However, some of them encode proteins with unknown functions. Characterization of these genes will potentially give additional opportunities to modify fibre properties. This thesis describes the discovery and characterization of a highly upregulated gene with a previously unknown function in poplar xylem, here denoted PttMAP20. Following its early discovery by mRNA profiling, the characterization was initiated with a thorough bioinformatic analysis, and the knowledge obtained was used to devise techniques for further functional analysis. Specific antibodies were raised, affinity purified and characterized. The antibodies were used as a tool for screening recombinant expression in E. coli and for the cellular localization of the protein in plant tissues, visualized with confocal and transmission electron microscopy. A purification protocol was developed for the expressed protein, followed by biochemical characterization. Appropriate model systems were used in both in vivo and in vitro studies. Fluorescently labelled protein transiently expressed in tobacco leaves was used for localization studies and the same system was used to characterize the molecular properties of the protein. Phenotypes arising from overexpressing the PttMAP20 gene were traced in the model plant Arabidopsis. All the results obtained so far indicate that PttMAP20 is a novel microtubule associated protein that binds to a cellulose biosynthesis inhibitor, DCB (2,6-dichlorobenzonitrile) and is required during cellulose biosynthesis in secondary cell walls. / QC 20100906

In previous studies the carboxyl-terminus of microtubule-associated protein 1S was shown to interact with the N-methyl-D-aspartate receptor subunit NR3A (Eriksson et. al.)1.  In this study, interactions between three truncations of the microtubule-associated proteins 1B and one truncation of the microtubule-associated protein 1S carboxyl-terminus and the N-methyl-D-aspartate receptor subunits NR1 and NR3A were examined. The study showed that an interaction occurred between amino acids 2167 to 2365 of the microtubule-associated protein 1B and NR3A.  That region of microtubule associated protein 1B corresponds to a microtubule-binding region in the light chain. It has been shown in earlier studies (Reviewed in Halpain S. et a12, Riederer, BM.  et.al3.) that the light chain is a active part of the protein that have been post translational cleaved. The MAP 1 proteins are present in all tissue but has higher concentrations in the Post Synaptic Density of neurons in the central nervous system.  The N-methyl-D-aspartate receptors are present in glial cells and in the dendritic shafts of the central nervous system neurons (Eriksson et. al.)1 . The diseases were these proteins may play a part is mainly memory destructive diseases such as Alzheimers disease and in muscular dystrophy, but these assumptions are still being speculated.