Cytoskeletal Mechanics and Mobility in the Axons of Sensory Neurons

The axon is a long specialized signaling projection of neurons, whose cytoskeleton is composed of networks of microtubules and actin filaments. The dynamic nature of these networks and the action of their associated motor and cross-linking proteins drives axonal growth. Understanding the mechanisms that control these processes is vitally important to neuroregenerative medicine and in this dissertation, evidence will be presented to support a model of interconnectivity between actin and microtubules in the axons of rat sensory neurons. First, the movement of GFP-actin was evaluated during unimpeded axonal outgrowth and a novel transport mechanism was discovered. Most other cargoes in the axon are actively moved by kinesin and dynein motor proteins along stationary microtubules, or are moved along actin filaments by myosin motor proteins. Actin, however, appears to be collected into short-lived bundles that are either actively carried as cargoes along other actin filaments, or are moved as passive cargoes on short mobile microtubules. Additionally, in response to an applied stretch, the axon does not behave as a uniform visco-elastic solid but rather exhibits local heterogeneity, both in the instantaneous response to stretch and in the remodeling which follows. After stretch, heterogeneity was observed in both the realized strain and long term reorganization along the length of the axon suggesting local variation in the distribution and connectivity of the cytoskeleton. This supports a model of stretch response in which sliding filaments dynamically break and reform connections within and between the actin and microtubule networks. Taken together, these two studies provide evidence for the mechanical and functional connectivity between actin and microtubules in the axonal cytoskeleton and suggest a far more important role for actin in the development of the peripheral nervous system. Moreover this provides a biological framework for the exploration of future regenerative therapies

Zebrafish as a Model for the Study of Live in vivo Processive Transport in Neurons

Motor proteins are responsible for transport of vesicles and organelles within the cell cytoplasm. They interact with the actin cytoskeleton and with microtubules to ensure communication and supply throughout the cell. Much work has been done in vitro and in silico to unravel the key players, including the dynein motor complex, the kinesin and myosin superfamilies, and their interacting regulatory complexes, but there is a clear need for in vivo data as recent evidence suggests previous models might not recapitulate physiological conditions. The zebrafish embryo provides an excellent system to study these processes in intact animals due to the ease of genetic manipulation and the optical transparency allowing live imaging. We present here the advantages of the zebrafish embryo as a system to study live in vivo processive transport in neurons and provide technical recommendations for successful analysis

Loss of Miro1-directed mitochondrial movement results in a novel murine model for neuron disease.

Defective mitochondrial distribution in neurons is proposed to cause ATP depletion and calcium-buffering deficiencies that compromise cell function. However, it is unclear whether aberrant mitochondrial motility and distribution alone are sufficient to cause neurological disease. Calcium-binding mitochondrial Rho (Miro) GTPases attach mitochondria to motor proteins for anterograde and retrograde transport in neurons. Using two new KO mouse models, we demonstrate that Miro1 is essential for development of cranial motor nuclei required for respiratory control and maintenance of upper motor neurons required for ambulation. Neuron-specific loss of Miro1 causes depletion of mitochondria from corticospinal tract axons and progressive neurological deficits mirroring human upper motor neuron disease. Although Miro1-deficient neurons exhibit defects in retrograde axonal mitochondrial transport, mitochondrial respiratory function continues. Moreover, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or mitochondrial calcium buffering. Our findings indicate that defects in mitochondrial motility and distribution are sufficient to cause neurological disease

Current methods to analyze lysosome morphology, positioning, motility and function

Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.Medical Biochemistr

Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion

The opportunistic pathogen Toxoplasma gondii is an obligate intracellular parasite part of the phylum Apicomplexa, able to infect all warm-blooded animals including humans. Invasion by apicomplexan parasites such as Plasmodium falciparum and Toxoplasma gondii to host cells requires the establishment and crossing through of a small ring-like junctional structure serving as an interface and stabiliser between the parasite and host cell plasma membrane. During the invasion process, the host cell possibly resist invasion to some degree, exerting force on the parasite’s entry point as de novo actin polymerisation has been characterised in this location (Gonzalez et al., 2009). Additionally, the parasite is required to generate force via an actomyosin motor to achieve host cell membrane penetration successfully, leading to mechanical deformation when the parasite is squeezing through the junctional ring. This actomyosin motor depends on a protein complex termed the glideosome, that pulls actin to achieve forward motility. Actin plays a key role in the parasite’s biology with important functions not only during invasion but also during replication, apicoplast maintenance and egress. Until recently, the lack of reliable F-actin sensors hampered the characterisation of actin dynamics during these processes. With the use of nanobodies with the potential to recognise actin (Periz et al., 2017), a complex actin behaviour was uncovered allowing the assessment of in vivo dynamics through the parasite’s lytic cycle. The uncovered flow of F-actin presented new opportunities to address debate over stablished hypothesis on parasite’s actin and to extend the initial roles attributed to actin including the establishment of cytoplasmic actin pool through the parasite’s life. Additionally, these F-actin dynamics were shown to be affected by traditional actin modulating drugs, as well as interference with actin binding factors resulting in abrogation of these dynamics and phenotypes associated with motility. Additionally in this thesis, it is suggested that F-actin’s role in invasion goes beyond powering the glideosome via force traction, but to facilitate nucleus passage and deformation. Real time and super resolution microscopy highlighted that during invasion events, the junction ring can oppose nucleus passage as parasites deficient of core components of the acto-myosin system have been shown to be incapable of withstand pressure exerted at the junction ring, leading to blebbing and collapse of the invading parasite (Bichet et al., 2016). Although some of these parasites are able to complete invasion, the dynamics are visibly affected suggesting more systems are at play during invasion. The literature shows that other eukaryotic systems deploy nucleus protection and displacement mechanisms to facilitate migration through tight spaces by the concerted action of actomyosin complexes and cytoskeletal structures (Petrie et al., 2012; Petrie and Yamada, 2015; McGregor, Hsia and Lammerding, 2016). This thesis proposes that the F-actin machinery facilitates nucleus passage through the junctional ring, offering a model fort the dual contribution of F-actin forces by constricting and pushing/pulling the nucleus during host cell invasion by these apicomplexan parasites, sharing similar mechanism with those of larger eukaryotes

Nucleoporin mRNA localization and Annulate Lamellae biosynthesis during Drosophila melanogaster oogenesis

Nuclear pore complexes (NPCs) are large protein assemblies that connect the eukaryotic nucleus with the cytoplasm, thus facilitating all transport between them. Besides the nuclear envelope (NE), NPCs also occur in parallel stacks of cytoplasmic membranes called Annulate Lamellae (AL) that can serve as storage, facilitating rapid nuclear growth via NE insertion during fruit fly embryogenesis. How and when AL are assembled is largely unknown. In this work, I established that AL are already abundant in late stage oocytes, and progressively accumulate throughout oogenesis specifically in the oocyte. By screening the localization of 39 nucleoporin and related mRNAs, I detected the specific enrichment of two nucleoporin and three importin encoding transcripts to AL, the NE, and previously unidentified nucleoporin granules throughout the egg chamber. Perturbation experiments revealed a dependence on active translation, but independence of an intact microtubule network on mRNA localization. Generation of GFP::Nup358 transgenic flies revealed granules with distinct partial nucleoporin contents, that are subject to microtubule-dependent transport and interactions among them. Their spatiotemporal abundance distribution is indicative of NPC precursors, and they contain partial accumulations of pore complexes within internal membranes. These granules further displayed characteristics of biomolecular condensates, including fast intra-granule dynamics, exclusion of cytoplasmic constituents, and sensitivity to 1,6-hexanediol. Both condensation state and AL assembly were dependent on Ran, a protein also fundamental for NPC assembly at the NE. Its nucleotide status throughout this is likely controlled by differential localization of its modulators RanGAP and Rcc1 to granules and cytoplasm respectively. This work thus established a molecular framework and basic sequence of events that leads to the assembly of AL, which are crucial during early development, and might have broader implications for NPC assembly also at the NE

An investigation into the transport and modulation of synaptophysin positive vesicles

Neuronal function, survival and architecture all critically depend on the precise transport of intracellular proteins to a vast array of synaptic connections. Disrupted intracellular transport leads to deficits in synaptic transmission, irregular cell morphology, misallocated organelles and cell death. In addition, axonal transport deficits have been noted in the early stages of several debilitating neurological conditions, thus, axonal transport deficits may contribute to disease progression. This makes it important that we understand the contribution of axonal transport to both physiological and pathophysiological cellular processes and to the transport of essential organelles. As such the aims of this project were as follows: to investigate the long-term transport properties of visualised synaptic vesicles, to investigate whether vesicle transport could be modulated by changes in neuronal activity, to examine whether vesicle transport deficits exist in certain disease models and to develop novel assays for focusing the study of vesicle transport to specific neuronal cell types. To investigate the transport properties of visualised synaptic vesicles we exploited a lentiviral vector to express a fluorescently tagged version of an abundant synaptic vesicle transmembrane protein, synaptophysin. Using synaptophysin-GFP (syp-GFP) as a synaptic vesicle marker we then tracked the movements of synaptic vesicles in the axons of dissociated hippocampal neurons. Synaptophysin-GFP expression revealed two fluorescent vesicle populations, one population that moved in a rapid and bi-directional manner and one population that accumulated into clusters of stationary vesicles at putative presynaptic sites. Each vesicle population was analysed independently. Moving vesicles were termed motile particles, whilst vesicle accumulations were termed vesicle clusters. To investigate potential activity-dependent changes in vesicle transport and vesicle cluster localisation we used acute or co-culture application of the GABAA receptor antagonists bicuculline (bic) (20µM) or Gabazine (gbz) (20µM), which can generate increased neuronal activity or epileptiform-like activity in vitro. As a result of bic treatment we observed a significant decrease in the size of stationary presynaptic vesicle clusters. Under control conditions the average size of vesicle clusters was 14.7±1.67µm2, reducing to 12.1±1.41µm2 following 10 hours of increased neuronal activity (p=0.0042, Wilcoxon-matched pairs test, n=80, 8 experiments). In addition, increased neuronal activity also led to a significant increase in vesicle cluster turnover, which increased from 28±6.89% under control conditions to 44±8.46% as a result of increased neuronal activity (p=0.0261, unpaired student t-test, n=25, 11 experiments). However, these changes were not accompanied by any alteration in vesicle transport, with the speed, the density and the proportion of motile particles remaining unaffected by increased neuronal activity (table 3.1). This suggests that each vesicle population may therefore be differentially modulated by increased neuronal activity. To probe deeper for potential activity-dependent vesicle transport changes we restricted our study of vesicle transport to a specific axonal subtype, the hippocampal mossy fiber. To visualise mossy fiber vesicle transport, lentivirus expressing syp-GFP was pressure injected directly into the cell body layer of the dentate gyrus (DG) in hippocampal organotypic slice cultures. This revealed syp-GFP positive vesicles occupying both small (2-15µm3) and large (˃15µm3) mossy fiber synaptic terminals, which were found in and along the stratum lucidum. By examining the distribution of vesicle clusters at different time points following gbz or bic treatment (0hrs, 4hrs, 12hrs, 24hrs and 48hrs) we were able to show that epileptiform activity caused a delayed (>12 hours) but significant decrease in the proportion of large vesicle clusters. By 24 and 48 hours there was a significant decrease in the proportion of large vesicle clusters following bic treatment, decreasing from 9.4±1.21% under control conditions (n=11, 5 experiments) to 4.84%±0.72% after 24hrs (n=10, 4 experiments) and to 3.3±0.73% after 48hrs (n=12, 5 experiments), P<0.001, one-way ANOVA. This decrease in the proportion of large vesicle clusters may represent an important pathophysiological change triggered by epileptiform activity. Importantly, we also observed the same decrease in the proportion of large vesicle clusters in a mouse model of Rett syndrome, which models a severe neurodevelopmental disorder caused by a mutation in the gene coding MeCP2. As a consequence of bic treatment we observed a significant decrease in the proportion of large vesicle clusters from 7.2% ±1.78% in control cultures (n=6, 2 experiments), down to 0.9% ±0.6% in 48hr bic treated cultures (n=8, 3 experiments) and recovering to 6.9%±1.5% following bic wash out (n=11, 3 experiments); p<0.0001, one way ANOVA. Interestingly, Mecp2Stop/y hippocampal organotypic slices showed a greater decrease in the proportion of large vesicle clusters following 48hrs of bic treatment. The proportion of large vesicle clusters in 48hr bic treated WT slices was 3.3%±0.73%, whilst in 48rs bic treated Mecp2Stop/y slices it was 0.9%±0.6%, p=0.01, two-way ANOVA. These observations suggest that Mecp2Stop/y hippocampal organotypic slices are more sensitive to epileptiform activity than WT slices and may possess deficits in the vesicle transport system. Primary dissociated hippocampal cell cultures benefit from being both optically and experimentally accessible but lack a defined cellular arrangement. This hampers both the identification and study of specific cell types and specific synaptic connections. To overcome this limitation we developed a modified dissociated cell culture assay for defining the arrangement of dissociated hippocampal neurons. We cultured purified DG and CA3 cell populations in close opposition using a magnetic barrier, but transduced only DG granule cells with lenti-synaptophysin-GFP in order to visualise vesicle transport specifically in mossy fibers. Immunocytochemistry and vital dyes were used to confirm that specific cell populations could be cultured in close proximity, to confirm that lentiviral transduction was highly selective to DG granule cells and to post-hoc identify that vesicle trafficking was occurring specifically in mossy fibers. Using this method it was possible to image vesicle transport specifically in mossy fibers and to investigate vesicle cluster dynamics at putative MF-CA3 synapses. We conclude that this method is a significant improvement to previous techniques because dissociated cells can be arranged to form physiologically relevant synaptic connections, whilst remaining highly accessible to both live imaging and experimental manipulation

Subcellular Molecular Profiling of Midbrain Dopamine Neurons

Midbrain dopamine neurons play a critical role in motor function, motivation, reward, and cognition by providing modulatory input to cortical and basal ganglia circuits. Given the importance of dopamine neurotransmission and its dysregulation in disease, mechanistic insight into the molecular underpinnings of dopaminergic neuronal function is needed. This thesis seeks to advance our understanding of dopamine neuronal cell biology by developing and applying cutting edge molecular profiling methods to study the subcellular translatome and proteome of dopamine neurons in mice. Chapter 1 provides an overview of the anatomy and cell biology of midbrain dopamine systems, with a particular emphasis on dopamine neurotransmission, neuronal heterogeneity, and selective vulnerability in Parkinson’s disease. Chapter 2 focuses on methods for studying local translation in neurons and describes newly discovered artifacts associated with two of these methods. Chapter 3 describes a global analysis of ribosome and mRNA localization in dopamine neurons; the results suggest that local translation in dopaminergic dendrites, but not axons, regulates dopamine release. Chapter 4 presents a method for subcellular proteomic profiling of dopamine neurons in the mouse brain, revealing the somatodendritic and axonal polarization of proteins encoded by Parkinson’s disease-linked genes. Emerging data are presented on Synaptotagmin 17, a novel axonal protein identified in midbrain dopamine neurons. Finally, I synthesize key findings regarding the molecular organization underlying dopamine neuronal cell biology and highlight promising areas for future investigation

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin