A study on morphological and dynamical properties of neuronal growth cones

In the developing nervous system and in the adult brain, neurons constantly need to solve mechanical problems. Neuronal growth cones are the main motile structures located at the tip of neurites and are composed of a lamellipodium from which thin filopodia emerge. They are responsible for extension of neurite processes and for transducing signals from extracellular cues to alter directionality, branching, and motility. They must decide how to explore the environment and in which direction to grow; they also need to establish appropriate contacts, to avoid obstacles and to determine how much force to exert. The complete understanding of the nervous system and its basic unit, the neuron, demands a quantification of the behavioral pattern and the morphological characteristics..

IST Austria Thesis

Blood – this is what animals use to heal wounds fast and efficient. Plants do not have blood circulation and their cells cannot move. However, plants have evolved remarkable capacities to regenerate tissues and organs preventing further damage. In my PhD research, I studied the wound healing in the Arabidopsis root. I used a UV laser to ablate single cells in the root tip and observed the consequent wound healing. Interestingly, the inner adjacent cells induced a division plane switch and subsequently adopted the cell type of the killed cell to replace it. We termed this form of wound healing “restorative divisions”. This initial observation triggered the questions of my PhD studies: How and why do cells orient their division planes, how do they feel the wound and why does this happen only in inner adjacent cells. For answering these questions, I used a quite simple experimental setup: 5 day - old seedlings were stained with propidium iodide to visualize cell walls and dead cells; ablation was carried out using a special laser cutter and a confocal microscope. Adaptation of the novel vertical microscope system made it possible to observe wounds in real time. This revealed that restorative divisions occur at increased frequency compared to normal divisions. Additionally, the major plant hormone auxin accumulates in wound adjacent cells and drives the expression of the wound-stress responsive transcription factor ERF115. Using this as a marker gene for wound responses, we found that an important part of wound signalling is the sensing of the collapse of the ablated cell. The collapse causes a radical pressure drop, which results in strong tissue deformations. These deformations manifest in an invasion of the now free spot specifically by the inner adjacent cells within seconds, probably because of higher pressure of the inner tissues. Long-term imaging revealed that those deformed cells continuously expand towards the wound hole and that this is crucial for the restorative division. These wound-expanding cells exhibit an abnormal, biphasic polarity of microtubule arrays before the division. Experiments inhibiting cell expansion suggest that it is the biphasic stretching that induces those MT arrays. Adapting the micromanipulator aspiration system from animal scientists at our institute confirmed the hypothesis that stretching influences microtubule stability. In conclusion, this shows that microtubules react to tissue deformation and this facilitates the observed division plane switch. This puts mechanical cues and tensions at the most prominent position for explaining the growth and wound healing properties of plants. Hence, it shines light onto the importance of understanding mechanical signal transduction

INVESTIGATING THE ROLES OF REACTIVE OXYGEN AND NITROGEN SPECIES IN PLANT PROGRAMMED CELL DEATH, CYTOSKELETAL AND MITOCHONDRIAL DYNAMICS

Mitochondria are usually considered simply as the “powerhouses of the cell”, however in recent years it has become apparent that mitochondria are also of fundamental importance in programmed cell death (PCD), which refers to cell death resulting from a controlled, genetically defined pathway. In Arabidopsis, PCD induced by either heat shock or treatment with strong oxidants is found to be correlated with an early and irreversible change in mitochondrial morphology which manifests as an increase in the size of individual mitochondria. In addition, PCD causes a clustering of mitochondria and loss of motility. In this study, I have used two arginase negative mutant Arabidopsis lines (argah1-1 and argah2-1) which have elevated cellular NO concentrations to examine the effect of nitrosative stress on mitochondria undergoing PCD. Another three different Arabidopsis lines (mito-GFP/mTalin-mCherry, mito-GFP/MAP4-mCherry, mito- mCherry/EB1b-GFP) were used to visualize cytoskeletal elements alongside mitochondria to examine the mechanisms responsible for the mitochondrial morphology transition, clustering and motility inhibition. Results indicate that the elevated concentration of NO found in arginase negative mutants is not sufficient to induce PCD. There was no significant mitochondrial morphology or dynamic change detected between arginase negative mutants and wild type plants, with or without a heat shock. Disruption of either actin or microtubule (MT) cytoskeletal elements leads to the formation of mitochondrial clusters, although they showed different cluster morphology and sizes. Mitochondrial clusters were observed to be moving along the remaining actin cables after a mild heat treatment or cytoskeletal depolymerizing drug treatment. Intact microtubules or MT plus ends visualized with EB1b did not show any interaction with mitochondria under normal conditions. However, after a mild heat stress, EB1b appeared to be associated with clusters of enlarged, possibly swollen mitochondria

TNO1 & VPS45: SNARE-associated proteins required for plant growth

Cellular trafficking of cargo vesicles at the trans-Golgi network (TGN) is required for multiple processes such as cell expansion, stress responses and hormonal transport in plants. Activity of membrane proteins known as SNAREs drives membrane fusion events. Associated proteins such as tethering factors and Sec1/Munc18 proteins aid the fidelity and efficiency of these fusion events by interacting with SNAREs. The TGN-localized SYP41/SYP61/VTI12 SNARE complex is required for vacuolar and secretory cargo trafficking. TNO1, a putative tethering factor, associates with SYP41 and is required for TGN membrane fusion dynamics and proper SYP61 localization. My dissertation research discovered a new role of TNO1 in auxin transport-related physiology. Mutants lacking TNO1 (tno1) display decreased gravitropic bending of plant organs, delayed lateral root emergence and increased sensitivity to natural auxin and a cell influx-specific synthetic auxin. Reduced auxin asymmetry at the tips of elongating lateral roots and gravistimulated primary root tips in the mutants confirms TNO1’s role in cellular auxin transport during these processes. Loss of TNO1 does not affect bulk endocytosis and arrival of membrane cargo at the TGN, suggesting a specific effect of TNO1 in auxin transport mechanisms by possibly affecting subcellular trafficking of auxin transporters. The root gravitropic defects led me to hypothesize that root growth movements would be defective in the tno1 mutants. I discovered that tno1 mutant roots display exaggerated rightward deviation from the growth trajectory (skewing), correlated with an enhanced left-handed root epidermal cell file rotation, when grown on slanted impenetrable growth media. tno1 mutants also behave differently from wildtype in studies investigating the effect of microtubule-disrupting drugs on root skewing and cell expansion show. This suggests that TNO1 might have a role in microtubule-associated mechanisms driving skewing and cell expansion, though a direct effect on MT array orientation was not observed. Altogether, this suggests TNO1’s role in both auxin transport and possibly MT-associated processes. I also investigated the effect of a point mutation in VPS45, a SYP41-associated Sec1/Munc18 protein. The mutant (Atvps45-3) displays dwarf phenotypes with highly reduced plant organ sizes and cell expansion. Mutant root hairs are short and thick compared to wildtype root hairs, suggesting defects in polarized cell expansion processes. The endocytic and secretory routes in Atvps45-3 plants seem unaffected suggesting a specific effect of the mutation on cell expansion. Taken together, these results add to the knowledge of SNARE-associated proteins at the TGN and how post-Golgi traffic mediates lateral root emergence, gravitropism, root movement and root hair expansion

The FTLD risk factor TMEM106B controls lysosomal trafficking and dendrite outgrowth

Frontotemporal dementia is the second most common neurodegenerative disease in people younger than 65 years. Patients suffer from behavioral changes, language deficits and speech impairment. Unfortunately, there is no effective treatment available at the moment. Cytoplasmic inclusions of the DNA/RNA-binding protein TDP-43 are the pathological hallmark in the majority of FTLD cases, which are accordingly classified as FTLD-TDP. Mutations in GRN, the gene coding for the trophic factor progranulin, are responsible for the majority of familiar FTLD-TDP cases. The first genome-wide association study performed for FTLD-TDP led to the identification of risk variants in the so far uncharacterized gene TMEM106B. Initial cell culture studies revealed intracellular localization of TMEM106B protein in lysosomes but its neuronal function remained elusive. Based on these initial findings, I investigated the physiological function of TMEM106B in primary rat neurons during this thesis. I demonstrated that endogenous TMEM106B is localized to late endosomes and lysosomes in primary neurons, too. Notably, knockdown of the protein does neither impair general neuronal viability nor the protein level of FTLD associated proteins, such as GRN or TDP-43. However, shRNA-mediated knockdown of TMEM106B led to a pronounced withering of the dendritic arbor in developing and mature neurons. Moreover, the strong impairment of dendrite outgrowth and maintenance was accompanied by morphological changes and loss of dendritic spines. To gain mechanistic insight into the loss-of-function phenotypes, I searched for coimmunoprecipitating proteins by LC-MS/MS. I specifically identified the microtubule-binding protein MAP6 as interaction partner and was able to validate binding. Strikingly, overexpression of MAP6 in primary neurons phenocopied the TMEM106B knockdown effect on dendrites and loss of MAP6 restored dendritic branching in TMEM106B knockdown neurons, indicating functional interaction of the two proteins. The link between a lysosomal and a microtubule-binding protein made me study the microtubule dependent transport of dendritic lysosomes. Remarkably, live cell imaging studies revealed enhanced movement of dendritic lysosomes towards the soma in neurons devoid of TMEM106B. Again, MAP6 overexpression phenocopied and MAP6 knockdown rescued this effect, strengthening the functional link. The MAP6-independent rescue of dendrite outgrowth by enhancing anterograde lysosomal movement provided additional evidence that dendritic arborization is directly controlled by lysosomal trafficking. From these findings I suggest the following model: TMEM106B and MAP6 together act as a molecular brake for the retrograde transport of dendritic lysosomes. Knockdown of TMEM106B and (the presumably dominant negative) overexpression of MAP6 release this brake and enhance the retrograde movement of lysosomes. Subsequently, the higher protein turnover and the net loss of membranes in distal dendrites may cause the defect in dendrite outgrowth. The findings of this study suggest that lysosomal misrouting in TMEM106B risk allele carrier might further aggravate lysosomal dysfunction seen in patients harboring GRN mutations and thereby contribute to disease progression. Taken together, I discovered the first neuronal function for the FTLD-TDP risk factor TMEM106B: This lysosomal protein acts together with its novel, microtubule-associated binding partner MAP6 as molecular brake for the dendritic transport of lysosomes and thereby controls dendrite growth and maintenance.Frontotemporale Demenz ist die zweithäufigste Form neurodegenerativer Erkrankungen bei Menschen unter 65 Jahren. Patienten leiden an Verhaltensauffälligkeiten und Sprach- sowie Artikulationsstörungen. Leider steht zurzeit keine wirksame medikamentöse Therapie zur Verfügung. Das pathologische Hauptmerkmal der meisten FTLD-Fälle sind zytoplasmatische Einschlüsse des DNA/RNA-bindenden Proteins TDP-43. Diese Fälle werden entsprechend als FTLD-TDP klassifiziert. Für einen Großteil der familiären FTLD-TDP Fälle sind Mutationen in GRN, dem für den Wachstumsfaktor Progranulin kodierenden Gen, verantwortlich. Die erste für FTLD-TDP durchgeführte genomweite Assoziationsstudie führte zur Entdeckung von genetischen Varianten im bis dato uncharakterisierten Gen TMEM106B. Diese Varianten sind mit einem erhöten Risiko an FTLD zu erkranken assoziiert. Initiale Studien in Zellkultur zeigten eine Lokalisierung des TMEM106B Proteins in Lysosomen, die Frage nach der neuronale Funktion des Proteins blieb allerdings bisher unbeantwortet. Auf diesen ersten Ergebnissen aufbauend untersuchte ich während meiner Dissertation die physiologische Funktion von TMEM106B in primären Ratten-neuronen. Ich konnte zeigen, dass endogenes TMEM106B auch in primären Neuronen in späten Endsosomen und Lysosomen lokalisiert ist. Beachtenswerterweise verminderte die Herunterregulierung (shRNA-vermittelter Gen-Knockdown) des Proteins weder das generelle Überleben der Neuronen noch die Level von anderen FTLD-assoziierten Proteinen, wie GRN oder TDP-43. Die Herunterregulierung von TMEM106B führte jedoch zu einem ausgeprägten Verlust von Dendriten in sich entwickelnden und ausgereiften Neuronen. Des Weiteren war die starke Beeinträchtigung dendritischen Wachstums und Aufrechterhaltung von einer morphologischen Veränderung und dem Verlust der Dornfortsätze begleitet. Um den Mechanismus dieser Phänotypen zu erklären, suchte ich nach TMEM106B coimmunopräzipitierenden Proteinen mittels Massenspektrometrie. Ich konnte das Mikrotubuli bindende Protein MAP6 als spezifischen Bindungspartner identifizieren und die Interaktion beider Proteine validieren. Hervorzuheben ist, dass die Überexpression von MAP6 in primären Neuronen den Effekt der Herunterregulation von TMEM106B auf die Dendriten kopierte und die Herunterregulation von MAP6 die dendritischen Verästelungen in TMEM106B depletierten Neuronen sogar wiederherstellen konnte. Diese Ergebnisse legen eine funktionelle Interaktion beider Proteine nahe. Die Verbindung zwischen einem lysosomalen und einem an die Mikrotubuli bindenden Protein brachte mich dazu, den Mikrotubuli abhängigen Transport von dendritischen Lysosomen zu untersuchen. Bemerkenswerterweise zeigten mittels Lebendzellmikroskopie erzeugte Aufnahmen eine erhöhte Bewegung dendritischer Lysosomen Richtung Zellsoma in TMEM106B depletierten Neuronen. Auch in diesem Kontext konnte die Überexpression von MAP6 den Effekt kopieren und die Herunterregulation von MAP6 den Effekt aufheben und somit die These einer funktionellen Interaktion festigen. Die MAP6 unabhängige Wiederherstellung des dendritischen Wachstums durch die Erhöhung des lysosomalen Transports in anterograder Richtung lieferte einen zusätzlichen Beweis dafür, dass das dendritische Wachstum direkt von lysosomalem Transport abhängt. Ausgehend von diesen Ergebnissen schlage ich folgendes Modell vor: TMEM106B und MAP6 wirken zusammen als molekulare Bremse für den retrograden Transport dendritischer Lysosomen. Die Herunterregulation von TMEM106B und die (wahrscheinlich dominant negative wirkende) Überexpression von MAP6 lösen diese Bremse und verstärken die retrograde Bewegung von Lysosomen. Daraufhin könnten der gestiegene Proteinumsatz und der Verlust von Plasmamembranbestandteilen zu einem Fehler im dendritischen Wachstum führen. Die Ergebnisse dieser Arbeit legen nahe, dass fehlerhafter, lysosomaler Transport in TMEM106B Risikoallelträgern zu einer Verstärkung der lysosomalen Fehlfunktion in Patienten mit GRN Mutation führt und dabei zur Krankheitsentwicklung beiträgt. Zusammengefasst habe ich die erste neuronale Funktion für den FTLD-TDP Risikofaktor TMEM106B entdeckt: Dieses lysosomale Protein wirkt zusammen mit seinem neuentdeckten, Mikrotubuli assoziierten Bindungspartner MAP6 als molekulare Bremse für den dendritischen Transport von Lysosomen und kontrolliert dadurch Wachstum und Aufrechterhaltung von Dendriten

Cytoskeleton and Cell Motility

The present article is an invited contribution to the Encyclopedia of Complexity and System Science, Robert A. Meyers Ed., Springer New York (2009). It is a review of the biophysical mechanisms that underly cell motility. It mainly focuses on the eukaryotic cytoskeleton and cell-motility mechanisms. Bacterial motility as well as the composition of the prokaryotic cytoskeleton is only briefly mentioned. The article is organized as follows. In Section III, I first present an overview of the diversity of cellular motility mechanisms, which might at first glance be categorized into two different types of behaviors, namely "swimming" and "crawling". Intracellular transport, mitosis - or cell division - as well as other extensions of cell motility that rely on the same essential machinery are briefly sketched. In Section IV, I introduce the molecular machinery that underlies cell motility - the cytoskeleton - as well as its interactions with the external environment of the cell and its main regulatory pathways. Sections IV D to IV F are more detailed in their biochemical presentations; readers primarily interested in the theoretical modeling of cell motility might want to skip these sections in a first reading. I then describe the motility mechanisms that rely essentially on polymerization-depolymerization dynamics of cytoskeleton filaments in Section V, and the ones that rely essentially on the activity of motor proteins in Section VI. Finally, Section VII is devoted to the description of the integrated approaches that have been developed recently to try to understand the cooperative phenomena that underly self-organization of the cell cytoskeleton as a whole.Comment: 31 pages, 16 figures, 295 reference