Dynactin is a multi-subunit complex which was initially identified in 1991 as an activator of cytoplasmic dynein-driven microtubule-based organelle motility in vitro. Although genetic studies also supported the involvement of both complexes in the same functional pathways in yeast, filamentous fungi, and Drosophila, none of these findings yielded significant insights into dynactin's mechanism of action. The full range of cytoplasmic dynein functions in vertebrate cells has also remained poorly understood, due, in large part, to the lack of a specific method of inhibition. The present thesis work was designed to investigate these issues through a study of the 50 kDa subunit of dynactin. As a first step (Chapter 1), I cloned mammalian p50 and characterized its expression at the tissue and subcellular levels. Rat and human cDNA clones revealed p50 to be a novel α-helix-rich protein containing several highly-conserved structural features including one predicted coiled-coil domain. Immunofluorescence staining of p50, as well as other dynactin and cytoplasmic dynein components in cultured vertebrate cells showed that both complexes are recruited to kinetochores during prometaphase and concentrate near spindle poles thereafter. These findings represented the first evidence for dynactin and cytoplasmic dynein co-localization within cells, and for the presence of dynactin at kinetochores. The second major phase of the thesis (Chapter 2) was focused on investigating dynactin and cytoplasmic dynein function in cultured cells in vivo using a dominant negative inhibition approach based on transient transfections of p50 constructs. Overexpression of wild type human p50 in cultured cells resulted in a dramatic fragmentation and dispersal of the Golgi apparatus. Time-lapse fluorescence microscopy analysis of p50-overexpressing cells revealed that microtubule-based vesicle transport from the endoplasmic reticulum to the Golgi was inhibited. Also, the interphase microtubule organizing center was found to be less well-focused in some but not all transfected cells. Overexpression of p50 also disrupted mitosis, causing cells to accumulate in a prometaphase-like state. Chromosomes were condensed but unaligned, and spindles, while still generally bipolar, were dramatically distorted. Sedimentation analysis revealed the dynactin complex to be dissociated in the transfected cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores was markedly diminished in cells expressing high levels of p50. These findings provided the first in vivoevidence for the role of dynactin in cytoplasmic dynein function, i.e. mediating the motor's binding to at least one "cargo" organelle, the kinetochore, and probably also to others such as vesicles destined for the Golgi complex. These data also strongly implicated both dynactin and dynein in Golgi organization during interphase, and chromosome alignment and spindle organization during mitosis. Based on the remarkable disruptive phenotypic effects associated with overexpressing of p50, the name of dynamitin was proposed for this polypeptide. In the third and last phase of the thesis (Chapter 3), two issues were addressed: first, the dynamitin-induced mitotic arrest phenotype was studied in greater detail to better understand the exact sites of dynactin and cytoplasmic dynein activity throughout mitosis. Second, a domain analysis of dynamitin was performed to gain insight into its function within the dynactin complex. A time-lapse fluorescence microscopy study of mitosis in living dynamitin-overexpressing COS-7 cells strongly suggested specific defects in interactions of astral microtubules with the cell cortex, and in both spindle pole assembly and maintenance. Analysis of the mitotic arrest phenotype in a second cell line revealed a second arrest point at metaphase, and a clear effect of dynamitin overexpression on spindle axis orientation, again consistent with defects in interactions between microtubules and the cell cortex. Refined analyses of kinetochore and spindle pole components also confirmed specific defects in kinetochore function and spindle pole organization. Taken together, these findings support three main sites of dynactin and cytoplasmic dynein activity during vertebrate mitosis: prometaphase kinetochores, spindle poles, and the cell cortex. Finally, the domain analysis revealed dynamitin to be capable of self-association through at least two separate interaction domains, consistent with models of the mechanism underlying dynamitin-induced dynactin dissociation, and therefore, yielding important new insights into dynactin assembly. This study also indicated that a third region within dynamitin, residues 105 to 154, is essential for dynamitin and dynactin function. An independent study confirmed this finding, implicating this region in binding to ZW10, an upstream kinetochore protein. Dynamitin has therefore been revealed to be the kinetochore-targeting subunit of dynactin, and indirectly, cytoplasmic dynein. Through the body of this thesis work, dynamitin has also emerged as a powerful new tool for studying vertebrate dynactin and cytoplasmic dynein function in vivo and in vitro.

In plant cells, actin filaments are nucleated in two different ways: The growth of single filaments or their bundles is enabled by various types of formins, whereas branched meshworks emerge due to Arp2/3 complex activity. Mutations in genes of these nucleators lead to various phenotypic traits. This thesis deals in the first place with influence of Arp2/3 complex subunits' dysfunction on intracellular motility (cytoplasmic streaming, stop-and-go movements of Golgi apparatus cisternae), since it had not been extensively studied before, and also attempts to quantify the already known impacts of mutations in genes for ARP2 and ARPC5 subunits on the vacuolar morphogenesis. For comparison, a few experiments with plants which carried a mutation in gene for FH1 formin were also realised when measuring the cytoplasmic streaming. The experiments were conducted with a model plant Arabidopsis thaliana. The methods particularly included transformation with fluorescent markers by Agrobacterium tumefaciens (or usage of a fluorescent dye), microscopy (both standard and confocal) and subsequent evaluation of the acquired data using a computer. During the cytoplasmic streaming research, effects of cytoskeletal drug latrunculin B were studied, too. The outputs did not prove that the Arp2/3 complex defects would manifest...