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Intermediate filaments in mammalian cell motility
Čermáková, Kateřina ; Libusová, Lenka (advisor) ; Pelantová, Markéta (referee)
Cell migration is crucial for the formation and maintenance of a multicellular mammalian organism, contributing to important processes such as embryonic development, tissue renewal, and immune surveillance. It is a complex phenomenon involving a plethora of processes, including relevant signalization. An impairment of those processes could be projected into innumerable pathological states, from wound healing disruption to tumour metastasis and invasiveness, thus the interest of many researchers has turned toward migration. The migration of mammalian cells is dependent on a cytoskeleton, which is being considerably rearranged in motile cells. This thesis aims to summarise the role of intermediated filaments in cell motility - the less understood cytoskeletal network in this context. Cytoplasmic, as well as nuclear intermediate filaments, due to their unique mechanical properties, affect cell mechanics. They protect against physical stresses (as cells squeeze through confined pores in a complex intercellular microenvironment) and modulate and direct actomyosin-generated forces, which are the main driving force of migration. In addition, they contribute to important migration- involved steps, such as cell polarisation, cell adhesion to surrounding surfaces, cohesion in collective migration, and...
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Vesicular roles of Arp2/3 nucleation-promoting factors
Dostál, Vojtěch ; Libusová, Lenka (advisor) ; Malínský, Jan (referee) ; Befekadu, Asfaw (referee)
F-actin is involved in key aspects of vesicular traffic, such as membrane deformation, tubulation and vesicle motion. Branching of F-actin is mediated by Arp2/3 but this complex must first be activated by so-called nucleation-promoting factors (NPFs). These factors play an essential role in the decision where and when branched actin should form on the membrane surface. The thesis focuses on the mechanisms which underlie localization and activation of NPFs, especially in terms of the phosphoinositide composition of the vesicle membranes. I show that one of the NPFs, the WASH complex, does not exclusively depend on the retromer complex for its membrane anchoring, as previously theorized. Rather, its understudied subunit SWIP enables the complex to independently bind to the membrane. I also present data showing that the WASH complex has essential roles in maintaining lysosomal function. Additionally, I elucidate the function of another NPF known as WHAMM in the ERGIC compartment, showing that it depends on the presence of myotubularin 9 for its ability to form membrane tubules. The thesis improves our understanding of the interface between the actin cytoskeleton and intracellular membrane system.
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The role of anillin in the growth cone of neurons
Tomášová, Štěpánka ; Libusová, Lenka (advisor) ; Vinopal, Stanislav (referee)
During embryonal development, axons of newly differentiated neurons need to properly interconnect and create a functional neuronal network. To achieve this, the cell requires a growth cone. The growth cone is a highly dynamic structure at the end of growing axons that serves both as the navigator and the propeller. Crosstalk between actin and microtubules is vital for proper axonal pathfinding. But the exact mechanism of this cooperation remains unknown. This diploma thesis investigates the possible role of a candidate scaffolding protein called anillin in this process. Anillin has been studied in two human cell lines. SH-SY5Y neuroblastoma cell line was used for overexpression and siRNA knock-down experiments. Anillin overexpression led to perturbed neurite morphology and growth cone dynamics in SH-SY5Y cells, whereas cells with lower anillin expression had fewer neurites. Next, neurons differentiated from human iPSC (induced pluripotent stem cells) expressing endogenous fluorescently tagged anillin were studied. Local dynamic high concentration spots of anillin have been observed at the base of cell protrusions of differentiating neurons. These anillin flares appeared during cell migration, early neurite initiation, and in newly created growth cones. These results suggest that anillin plays a...
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Role of NAV3 in glioblastoma cells invasiveness
Legátová, Anna ; Brábek, Jan (advisor) ; Libusová, Lenka (referee)
The invasion of tumor cells from the primary lesion and the formation of metastases are the main reasons for the severe impact of cancer diseases. An option for dealing with this poor impact is the development of drugs (so-called migrastatics) that would target these processes and thus limit the spread of tumor cells from the site of the primary tumor. However, to develop such drugs, it is essential to clarify the molecular mechanisms that control or promote cell migration. One of the possible strategies for migrastatics development is the targeting of cytoskeletal structures, which play an indispensable role in cell migration. This work is focused on Neuron navigator 3 (NAV3), a protein that binds to + ends of microtubules (MTs), participates in their stabilization, and is able to mediate crosstalk between MTs and the actin network. The function of NAV3 is important for directing MTs into growing axons and proper neurite outgrowth, which is necessary for brain development. The results of this thesis suggest that NAV3 could act as a pro-tumor factor, which localizes not only to the + ends of MTs, but also to the cell protrusions, and whose presence supports the cell expansion and increases the invasive potential of glioblastoma cell lines. Key words: neuron navigator 3, microtubules, cancer,...
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The role of the WASH complex in endolysosomal homeostasis
Beránková, Pavla ; Libusová, Lenka (advisor) ; Rösel, Daniel (referee)
The WASH (WASP and SCAR homologue) complex is an actin nucleation promoting factor essential for endosomal cargo sorting. Upon WASH complex depletion, endosomal cargoes are mislocalized and the endolysosomal system collapses. Here, we employed high-speed vesicle tracking and real-time rescue experiments to test the effect of WASH complex depletion on endolysosomal homeostasis. We found that large lysosome-like vacuoles emerge in knockout cell lines of individual WASH complex subunits, although the overall dynamics of the lysosomal network does not substantially change. Follow-up experiments revealed that the WASH complex does not act directly on the vacuoles during their rescue. Overall, the data indicate that the emergence of vacuoles in WASH complex knockouts is a secondary process that depends on the WASH complex indirectly.
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Cytoskeletal orchestration of early mammalian development
Novotný, Jan ; Libusová, Lenka (advisor) ; Mašek, Jan (referee)
Structures of the cytoskeleton play a critical role in early mammalian development. Microtubules, intermediate filaments and actin microfilaments provide structural and mechanical basis for major developmental transitions of preimplantation development. Microvilli supported by bundles of actin filaments become restricted to one pole of the blastomere during the process of polarisation. All components of the cytoskeleton network undergo apparent reorganisation in embryo compaction. Cells flatten and adhere to each other thanks to the formation of cytoskeleton-linked intercellular junctions and oscillatory contractions generated by actomyosin complexes. Filopodia, actin based protrusions, are debated for their potential role in blastomere apposition and cell signalling. The first process of cell line specification in mammalian development is that which defines outer cells of morula as presumptive trophectoderm and inner cells as presumptive inner cell mass. Proteins and structures of cytoskeleton are asymmetrically distributed between these two populations helping to determine the fate of each blastomere. The aim of the thesis is to summarise the roles of cytoskeleton in blastomere polarisation, embryo compaction and specification of inner and outer cell populations in mammalian preimplantation...
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The role of cytoskeleton in movement and navigation of neuronal growth cones
Olekšák, Adam ; Libusová, Lenka (advisor) ; Weissová, Romana (referee)
Growth cone is a dynamic structure localized at the tips of growing neurites. It detects guidance cues in the extracellular environment and enables growing neurite to properly respond to them. Cytoskeleton plays a key role in navigation of growing neurite. Microtubules and microfilaments enable the motion of the growth cone by generating force acting on the cytoplasmic membrane as well as on the substrate on which the neuron grows. Microfilaments are located mainly in the periphery of the growth cone. They are more dynamic than microtubules, and their polymerization and depolymerization enable formation of dynamic processes on the growth cone. These are used by neurons to gain information about their surroundings and for formation of adhesions on permissive substrates. Adhesions facilitate the growth of neurites. Direction, timing and speed of formation of the dynamic processes are regulated by a number of actin-associated proteins. These proteins are common targets of signalling pathways that are induced by activation of growth cones' cytoplasmic membrane-bound receptors by guidance cues. Along the microfilament bundles, microtubules enter the periphery of the growth cone aided by crosslinking and motor proteins. Microtubules are necessary for reducing the growth cone dynamics, the first step in...
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Regulation of epithelial plasticity by ERK1 and ERK2 isoforms
Rasl, Jan ; Vomastek, Tomáš (advisor) ; Rösel, Daniel (referee) ; Libusová, Lenka (referee)
The ERK pathway is an evolutionarily conserved three-tier signaling cascade comprised of protein kinases Raf, MEK, and ERK. These core kinases are arranged in a hierarchical order and the signal is transduced from Raf to MEK to ERK. The ERK pathway is activated by diverse extracellular signals and in response regulates many cellular processes including cell proliferation, differentiation, apoptosis, migration or epithelial plasticity. Given the role of the ERK pathway in regulating such fundamental cellular processes, the ERK pathway signaling is tightly controlled and its dysregulation has pathological consequences such as cancer development and progression. Although much is known about mechanisms underlying the signal transduction by the ERK signaling pathway, much less is known about how two highly homologous ERK1 and ERK2 isoforms contribute to the signaling by this pathway. In this thesis, I studied isoform-specific functions of ERK1 and ERK2 using epithelial Madin- Darby Canine Kidney (MDCK) cells overexpressing either ERK1 or ERK2. Obtained data show that overexpression of ERK2, but not ERK1, had significant effects on the morphology and functional phenotype of MDCK cells. Both ERK1 and ERK2 expressing cells were able to form cohesive clusters, but the only ERK2 overexpression affected...
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Roles of tubulin post-translational modifications in regulation of microtubule-based processes
Šliková, Pavlína ; Novák, Petr (advisor) ; Libusová, Lenka (referee)
Microtubular cytoskeleton plays crucial roles during diverse cellular processes, such as intracellular transport, cell motility and chromosome segregation during cytokinesis. Tubulin, the building block of microtubules, undergoes numerous post-translational modifications which affect microtubular dynamics and organization as well as their interaction with associated proteins. Understanding the role post-translational modifications play in the diversification of functions and properties of microtubules is key for our comprehension of the dynamics of the complex microtubule cytoskeleton. However, mechanisms behind the effect of post-translational modifications on microtubule cytoskeleton are not fully understood. In this work, we focus on the influence of post-translational modifications on microtubule polymerization and interaction with molecular motor kinesin-1. Using total internal fluorescence and interference reflection microscopy techniques, we here show that high levels of post-translational modifications on microtubules decrease the time of microtubule-kinesin interaction whereas binding affinity and median velocity are not significantly different on modified and unmodified microtubules. Further, we show that the absence of polyglutamylation on tubulin isotypes leads to a faster microtubule...
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Apoptosis of pancreatic β-cells induced by endoplasmic reticulum stress and its mechanisms in type 2 diabetes
Glatzová, Daniela ; Němcová, Vlasta (advisor) ; Libusová, Lenka (referee)
Increasing incidence of type 2 diabetes represents one of the principal threats to human health in the 21st century. Strong evidence indicates that the rise in incidence of type 2 diabetes is correlated with increasing levels of obesity and that important factor playing role in the development of this disease is an elevation in circulating glucose and fatty acids. Chronically increased concentration of these nutrients was shown to induce apoptosis of pancreatic beta-cells that subsequently contributes to diabetes progression. Despite intensive research, molecular mechanisms underlying this beta-cells loss are still unclear. However, there is increasing evidence that one of the key processes involved in glucose and fatty acid-induced beta-cell death is induction of endoplasmic reticulum stress. The aim of this work is to summarize the recent knowledge about induction of apoptosis by endoplasmic reticulum stress in pancreatic beta-cells in relation to type 2 diabetes.
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