Browsing by Subject "Cytoskeleton"

The bacterial protein FtsZ plays a vital role in cytokinesis in prokaryotes as it polymerizes to form an FtsZ ring (Z ring) at the division septum midcell. FtsZ exhibits a GTP hydrolysis activity and attempts have been made to model the kinetics of this process. There is a major discrepancy, however, over the concentration of GTP needed for activity. The dissociation constant (KD) between wild-type FtsZ protein and GTP was measured to be 30 nM using isothermal titration calorimetry. In contrast, several research groups have reported that GTP hydrolysis required GTP concentrations in the millimolar range. They used Michaelis-Menten kinetics to model the GTP concentration dependence and obtained an apparent binding constant (Km) in the range of 300-1,000 µM GTP. Km and KD are not identical measures of binding given that they differ by the kinetic constant governing catalysis, kcat, but we suggest that a five order of magnitude difference between the values is unprecedented and this was a problem that needed investigating.

My overall goal in this work has been to perform a comprehensive in vitro study of the FtsZ GTP hydrolysis activity using an enzyme-coupled regenerating system. With this system rates of GTP hydrolysis by FtsZ are obtained spectrophotometrically. I have confirmed for wild-type FtsZ that GTP hydrolysis rates show little to no dependence on GTP concentrations in the range of 50-3,000 µM, contradicting the high values of Km reported in some previous studies. Since we have failed to reproduce the high Km with three different preparations of FtsZ protein, we cannot propose a definitive mechanism for the previous results.

I also measured the GTP hydrolysis of several mutants of FtsZ: E238A, L169R, and FtsZ84 (G105S). I investigated each of these mutants to see if they had a high apparent Km. FtsZ84 had a low overall hydrolysis rate, but did show a large increase in hydrolysis rates when GTP was increased from 50-3,000 µM. We hypothesize that a lower affinity for GTP is not a Michaelis-Menten Km, but likely a reflection of a weak binding of GTP by FtsZ84, giving KD in the millimolar range.

Synapses – the delicate connections between our neurons – adjust and refine their strength to shape our brains, our thoughts, and our memories. Proteomic and genetic techniques have revealed that this process, known as synaptic plasticity, is tightly controlled by signaling cascades that ultimately expand or contract actin networks within postsynaptic sites. In this dissertation, I advance the field of synaptic plasticity by focusing on presynaptic terminals, which are equal partners with their postsynaptic counterparts. To date, the study of presynaptic plasticity has been difficult due to the limited number of presynaptic signaling molecules currently identified (particularly those regulating the cytoskeleton), as well as the lack of tools to manipulate these molecules specifically within presynaptic terminals. I therefore developed new experimental approaches to tackle both of these hurdles. After mapping presynaptic cytoskeletal signaling pathways in the mouse brain, I discovered a new mechanism of presynaptic plasticity that is driven by action potential-coupled actin remodeling.

Presynaptic terminals cannot be biochemically purified away from postsynaptic sites. This has restricted previous presynaptic proteomic studies to isolated synaptic vesicles or other fractions, which have only identified a few actin signaling molecules. I thus turned to a new proteomic method called in vivo BioID. This approach is based on proximity-based biotinylation, which labels proteins in a compartment of interest as defined by a bait protein. My choice of presynaptic bait worked beautifully, leading to the mass spectrometry-based identification of 54 cytoskeletal regulators, most of which were previously not known to be presynaptic. The networks of presynaptic actin signaling molecules turn out to be just as richly diverse as those of the postsynapse. Many proteins also converge on a Rac1-Arp2/3 signaling pathway that leads to the de novo nucleation of branched actin filaments. This reveals that the presynaptic cytoskeleton consists of a dynamic, branched actin network.

This finding was unexpected because Rac1 and Arp2/3 have long-established roles in the development and plasticity of the postsynapse. This also makes it difficult to isolate the presynaptic functions of these proteins. I thus created optogenetic tools and electrophysiological strategies to acutely and bidirectionally manipulate their activity specifically within presynaptic terminals. I showed that presynaptic Rac1 and Arp2/3 negatively regulate the recycling of synaptic vesicles, thereby driving a form of plasticity known as short-term depression. I also showed that this mechanism is conserved between excitatory and inhibitory synapses, demonstrating it is a fundamental aspect of presynaptic function. Finally, I conducted a series of experiments using two-photon fluorescence lifetime imaging (2pFLIM) with a FRET-based biosensor of Rac1 activity. I discovered that calcium entry during action potential firing activates Rac1 within presynaptic terminals. This establishes a new mechanism of short-term depression that is driven by an action potential-coupled signal to the presynaptic cytoskeleton.

This dissertation thus combines proteomics, optogenetics, electrophysiology, and 2pFLIM-FRET to gain new insights into presynaptic plasticity. These findings have three significant implications. First, they challenge the prevailing view that the Rac1-Arp2/3 pathway functions largely at excitatory postsynaptic sites. This compels re-evaluation of how mutations in Rac1 and Arp2/3 cause neurological diseases such as intellectual disability and schizophrenia. Second, the genetic and optogenetic tools I developed are the first way to specifically modulate short-term depression, finally allowing for the exact functions of this form of plasticity to be determined in vivo. This has particular relevance for working memory, which has been theorized to be controlled by short-term presynaptic plasticity. Finally, this study provides a proteomic framework and blueprint of experimental strategies to conduct a systematic genetic analysis of the presynaptic cytoskeleton, which may finally unify the controversial theories about presynaptic actin function. In sum, the experimental strategies and resources that I developed highlight the multifaceted, sophisticated signaling that occurs in presynaptic terminals. This may yet shed light on how we remember our experiences, and why we are who we are.

The ability of cells to migrate is of fundamental importance to a diverse array of biological processes, both physiological and pathological, such as development, the immune response and cancer cell metastasis, to name a few. The process of cell movement is a complicated cycle of coordinated steps involving dynamic and precise rearrangement of the actin-myosin cytoskeleton. As a critical component of the migration machinery, the molecular motor protein nonmuscle myosin II (myosin II) has long been a subject of scientific inquiry. It is now generally accepted that the contractile forces generated by myosin II contribute directly or indirectly to every step in migration. Interestingly, three isoforms of myosin II (myosin IIA, IIB and IIC) have been identified, and although each isoform performs the same basic molecular functions, recent findings suggest that the different myosin II isoforms make unique contributions to the motile process. In this dissertation work I used RNA interference technology to specifically deplete cells of myosin IIA and IIB in order to characterize the distinct migration phenotypes associated with loss-of-function of each individual isoform. Surprisingly, I found that the two myosin II isoforms perform not only distinct but opposing functions in cell migration, with myosin IIA and IIB normally inhibiting and facilitating proper cell movement, respectively. Furthermore, using pharmacological and microscopy techniques, I investigated the cellular mechanisms allowing for isoform-specific function. My results provide evidence for at least two isoform-specific regulatory mechanisms, namely selectivity in signaling pathways and subcellular distribution. A particularly significant finding is the identification of the different assembly properties of myosin IIA and IIB as the key element responsible for directing isoform-distinct distribution. Together the data presented herein represent a considerable advance in our understanding of the distinct functions and regulation of myosin II in cell motility.

Cell sheet morphogenesis is essential for metazoan development and homeostasis, contributing to key developmental stages such as neural tube closure as well as tissue maintenance through wound healing. Dorsal closure, a well-characterized stage in Drosophila embryogenesis, has emerged as a model for cell sheet morphogenesis. Closure is a remarkably robust process where coordination of conserved gene expression and signaling cascades regulate cellular movements that drive closure. While well-characterized, new ‘dorsal closure genes’ continue to be discovered due to advances in microscopy and genetics. Here, we use live imaging and a set of large deletions, deficiencies (Dfs), that together remove 98.9% of the genes on 2L in order to identify regions of the genome required for normal closure. We successfully screened 96.1% of the genes on 2L and identified diverse dorsal closure defects in embryos homozygous for 47 Dfs, 26 of which have no known dorsal closure gene located within the Df region. We have already identified pimples, odd-skipped, paired, and sloppy-paired 1 as dorsal closure genes on the 2L affecting lateral epidermal cell shapes, and anticipate we will continue to identify novel ‘dorsal closure genes’ with further analysis. We also investigate the changes in dorsal closure dynamics and forces in the even-skipped (eve) mutant, which has aberrant cell shapes and behaviors as well as reduced actin and myosin at the purse string, but completes closure. We find that loss of wg/wnt-1 signaling in eve causes the observed defects in closure and that crumbs, a regulator of actin and myosin, is mis-expressed. Additionally, laser microsurgery demonstrates that the eve or wg mutant embryos are under a global tension in the anterior-posterior direction. Lastly, we identify a lesion in echinoid that is responsible for the jagged purse string and ectopic zipping dorsal closure phenotype previously thought to be due to a lesion in Zasp52.

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen and is the leading cause of preventable blindness worldwide. Chlamydia is particularly intriguing from the perspective of cell biology because it is an obligate intracellular pathogen that manipulates host cellular pathways to ensure its proliferation and survival. This is achieved through a significant remodeling of the host cell’s internal architecture from within a membrane-bound vacuole, termed the inclusion. However, given a previous lack of tools to perform genetic analysis, the mechanisms by which Chlamydia induces host cellular changes remained unclear. Here I present genetic and molecular mechanisms of chlamydial manipulation of the host cytoskeleton and organelles. Using a forward genetics screen, InaC was identified as a necessary factor for the assembly of an F-actin structure surrounding the inclusion. InaC associated with the vacuolar membrane where it recruited Golgi-specific ARF-family GTPases. Actin dynamics and ARF GTPases regulate Golgi morphology and positioning within cells, and InaC acted to redistribute the Golgi to surround the Chlamydia inclusion. These findings suggest that Chlamydia places InaC at the inclusion-cytosolic interface to recruit host ARF GTPases and F-actin to form a platform for rearranging intracellular organelles around the inclusion. The inclusion is also surrounded by the intermediate filament vimentin and the chlamydial protease CPAF cleaves vimentin in vitro. CPAF-dependent remodeling of vimentin occurred selectively in late stages of the infection. In living cells, this cleavage occurred only after a loss of inclusion membrane integrity, suggesting that CPAF cleaves intermediate filaments specifically during chlamydial exit of host cells. In summary, I have implemented recent forward and reverse genetic approaches in Chlamydia to reveal how it employs effector proteins to manipulate the internal organization of cells in novel ways.

Microtubules often adopt non-centrosomal arrays in differentiated tissues, where they are important for providing structure to the cell and maintaining polarity. Although the formation and organization of centrosomal arrays has been well-characterized, little is known about how microtubules form non-centrosomal arrays.

In the mouse epidermis, centrosomes in differentiated cells lose their microtubule-anchoring ability through the loss of proteins from the centrosome. Instead, microtubules are organized around the cell cortex. The cell-cell adhesion protein desmoplakin is required for this organization. Our model is that desmoplakin recruits microtubule-anchoring proteins like ninein to the desmosome, where they subsequently recruit and organize microtubules.

To test this model, we confirmed that the microtubule-binding proteins Lis1, Ndel1, and CLIP170 are recruited by desmoplakin to the cell cortex. Furthermore, by creating an epidermis-specific conditional Lis1 knockout mouse, I found that Lis1 is required for cortical microtubule organization. Surprisingly, however, Lis1 is also required for desmosome stability. This work reveals essential desmosome-associated components that control cortical microtubule organization and unexpected roles for centrosomal proteins in epidermal function.

Although Lis1 is required for microtubule organization, it is not sufficient. I created a culture-based system to determine what other factors may be required for cortical organization for microtubules. My work reveals that stabilization of the microtubules is sufficient to induce their cortical organization. Functionally, cortical microtubules are important for increasing the mechanical integrity of cell sheets by engaging adherens junctions. In turn, tight junction activity is increased. Therefore, I propose that cortical microtubules in the epidermis are important in forming a robust barrier by cooperatively strengthening each cell-cell junction.

To determine whether desmosomes play similar roles in simple epithelia as stratified epithelia, I examined intestinal epithelial-specific conditional desmoplakin conditional knockout mice. Unexpectedly, I found that desmoplakin is not required for cell-cell adhesion and tissue integrity in the small intestine. Furthermore, it does not organize intermediate filaments. Desmoplakin is required, however, for proper microvillus architecture.

Overall, my studies highlight novel tissue-specific roles for desmosomes, in particular desmoplakin, in organizing and integrating different cytoskeletal networks. How desmoplakin's function is regulated in each tissue will be a new interesting area of research.

Many microbial pathogens have evolved to replicate within host cells. While a number of these pathogens reside within vacuolar compartments, others escape from host endosomal pathways to replicate intracytosolically. To counter microbial invasion, host cells employ numerous defense proteins to limit microbial growth and mediate pathogen destruction. Among these host defense proteins are a number of dynamin-like GTPases expressed in response to the cytokine Interferon-gamma, including the p65 Guanylate Binding Proteins (GBPs). Murine GBPs have previously been shown to target both vacuolar and cytosolic pathogens to mediate pathogen destruction and potentiate host inflammatory responses via both the canonical (caspase-1) and noncanonical (caspase-11) inflammasomes. However, whether these functions are conserved among the human orthologs of murine GBPs has remained unclear.

To determine whether the ability to physically target pathogens is conserved among the human GBPs, I monitored the localization of all seven human GBPs within cells infected with the cytosol-resident Gram-negative bacterium Shigella flexneri, the causative agent of bacillary dysentery. Among the human GBP paralogs, I identified the unique ability of GBP1 to physically associate with S. flexneri, and showed that GBP1-targeting extends to a second cytosolic Gram-negative bacterium, Burkholderia thailandensis, but not to the cytosolic Gram-positive bacterium Listeria monocytogenes. Using mutational analysis, I determine that GBP1 targeting is directed by a C-terminal Polybasic Motif (PBM) centered around three arginine residues, and further relies on a lipidated CaaX motif and protein oligomerization via the GBP1 Large GTPase domain. Among the human GBP paralogs, the combination of a PBM and CaaX motif is unique to GBP1. Furthermore, I found that rough lipopolysaccharide (LPS) mutants of S. flexneri co-localize with GBP1 less frequently than wildtype S. flexneri, suggesting that host recognition of O-antigen promotes GBP1 targeting to Gram-negative bacteria. GBP1-targeting to S. flexneri led to co-recruitment of four additional human GBP paralogs (GBP2, GBP3, GBP4, and GBP6).

S. flexneri and a number of other cytosolic bacteria promote bacterial dissemination by hijacking host actin cytoskeleton machinery to form actin comet tails which emanate from one pole of the bacterium and provide mechanical force to propel bacterium-containing extensions into neighboring cells. I found that while GBP1-targeted bacteria remain viable, they replicate within intracellular aggregates and fail to form actin comet tails. Accordingly, wildtype but not a PBM-deficient GBP1 mutant restricts S. flexneri cell-to-cell spread in plaque assays. I also found that S. flexneri counters GBP1-mediated host defenses using a secreted effector, IpaH9.8. Accordingly, human-adapted S. flexneri, through the action of IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillus B. thailandensis.

Finally, I examined the role of human GBP1 in shaping the host cell transcriptional response in S. flexneri infected cells, and found that GBP1 promotes the expression of several chemokines, including CXCL1, CXCL9, CXCL10, and CCL2, which act as chemoattractants for professional immune cells. This role in chemokine expression was independent of the GBP1 PBM and CaaX motif necessary for bacterial targeting, and extended not only to B. thailandensis, but also L. monocytogenes, which is untargeted by GBP1. Furthermore, GBP2 could functionally substitute for GBP1 to support expression of CXCL10, implicating other GBPs in the process.

Together, the work encompassed in this dissertation sheds light on the role of the human GBPs in host cell defense against intracellular pathogens, and identifies previously unknown roles for the GBPs in precluding bacterial actin-based motility and shaping the host transcriptional response to pathogens.