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SammanfattningSyftet med denna studie var att jämföra NADH-kopplade mätsystem och in vitro motilitets-analys (IVMA) för att bestämma aktiviteten hos isolerat myosin. Från NADH-kopplade analysmätningar bestämdes tre parameter: den maximala hastigheten med vilken myosin hydrolyserar ATP i frånvaro av F-aktin (V0), den maximala ATPas-hastigheten för myosin i närvaro av mättande aktin (kcat) och den koncentration av aktin som behövs för att nå halv maximal aktivering av myosin ATPas-aktivitet (KATPas). Från in vitro-motilitets-analys (IVMA) bestämdes två parametrar: fraktion av rörliga filament (FMF) och totala antalet rörliga filament (TMF). Från detta kunde vi uppskatta den fraktion av aktiva huvuden i myosinpreparationer som behövs för en lyckad IVMA.Myosin är ett protein som tillsammans med aktin är ansvarigt för muskelkontraktionen. I denna studie används två myosin preparationer (HMM-fragment) som vi betecknade ”bra HMM” och ”dåligt HMM” på grund av deras kvalitet för aktin motilitet. Först mättes ATPas-aktiviteten hos myosinmotorer med hjälp av ett NADH-kopplat mätsystem som bygger på övervakning av förändringen i absorbans av NADH. Därefter bestämdes V0, kcat, och KATPas för aktin-beroende av myosin-ATPast genom att mäta myosinaktivitet vid olika aktinkoncentrationer, följt av anpassning av data till Michaelis-Menten ekvationen.Parallellt utfördes IVMA-studier genom att HMM immobiliserades på ett objektglas som derivatiserats med trimetylklorsilan. Sedan observerades när HMM flyttar fram fluorescensmärkta aktinfilament i närvaro av ATP. Under samma förhållanden gav resultaten för basalt myosin ATPas aktivitet V0 värden som var ~0,03 ATP s-1 myosinhuvud -1for både dåligt och bra HMM. I en jämförelse mellan de två HMM vid olika F-aktin-koncentrationer var hastighet i ATP-förbrukningen högre för bra än för dåligt HMM. Anpassning av data till Michaelis-Menten-ekvationen gav kcat på 7,18 ATP s-1myosinhuvud-1för dåligt HMM jämfört med 11,21 ATP s-1 myosinhuvud-1för bra HMM (35 % högre). KATPas (Km) för dåligt HMM var lite högre jämfört med den för bra HMM. Vid IVMA-studierna var FMF och TMF 80 % respektive 98 % lägre för dåligt än bra HMM. Slutsatsen var att de två metoderna karakteriserar HMM-funktionen på olika sätt och med olika känslighet. Om man antar att bra HMM har nästan 100 % aktiva huvuden och eftersom man vet att uppmätt kcat är direkt proportionellt mot antalet aktiva myosinhuvuden ser man från dessa mätningar att mycket mer än 65 % av totalt myosin måste vara aktivt för att ge god aktinmotilitet i en IVMA.

Platelets play a vital role in processes of hemostasis and thrombosis under physiological and pathological conditions. Following vascular damage, platelets will accumulate and stably adhere to exposed subendothelial matrixes. The binding of platelet surface receptor Glycoprotein VI (GPVI) to exposed collagen initiates a signaling cascade, which culminates in platelet activation. Stimulation of GPVI pathways results in the generation of thromboxane and causes the platelets to secrete their granule contents. This generated thromboxane as well as constituents released from dense granules such as ADP, and serotonin, play an essential role in potentiating the platelet response through activation of other surface receptor mediated pathways. Importantly, downstream of all these separate pathways, kinases become activated and play a crucial role in phosphorylating their substrates to elicit critical cellular responses. Previously published studies have established the importance for myosin kinase in its role for phosphorylating the myosin light chain (MLC) downstream of ADP receptors. These studies have shown MLC phosphorylation occurs rapidly and is essential for shape change following the stimulation of ADP receptors. Technological advances in antibody development have resulted in the generation of commercially available phospho-specific antibodies for MLC phosphorylated on either threonine (Thr) 18 or serine (Ser) 19. These antibodies allowed us to revisit these prior studies and address whether phosphorylation on MLC (Ser) 19 would elicit one response while phosphorylation on (Thr) 18 may result in another functional response. Our result show, that MLC is phosphorylated rapidly on (Ser) 19 and plays an important role in shape change downstream of Gq pathways, while MLC (Thr) 18 phosphorylation occurs at a slower rate downstream of G12/13 pathways and contributes to platelet dense granule secretion. Protein kinase C's (PKC) are serine/threonine kinase, which become activated following the stimulation of many of the platelet surface receptors. PKCs are classified into three groups, classical (α, βI, βII, γ), novel (δ, ε, η, θ), and atypical (ζ, ι, λ, μ) based on their cofactor requirements for activation. The classical PKCs, which require diacylglycerol and calcium for their activation were investigated using the specific inhibitor Go6976. Much to our surprise, we demonstrated that downstream of GPVI pathways, Go6976 caused non-selective inhibition of Spleen tyrosine kinase (Syk) activity. This inhibition of Syk activity resulted in a concentration-dependent reduction in phosphorylation of downstream molecules Lat and PLCγ2 as well as platelet aggregation and secretion. Stimulation of surface receptors GPVI, CLEC-2, GPIb, and FcRIIa, all lead to the activation of tyrosine kinase pathways. The role for Syk in these pathways is essential and in the absence of its activity these pathways are completely shut down. We inadvertently discovered dextran sulfate (DxS) actives platelets. Our results show that DxS activates a Src-dependent pathway which does not utilize surface receptors GPVI, CLEC-2, GPIb, or FcRIIa. Platelets pretreated with Syk inhibitors OXSI-2 or Go6976 failed to cause αIIbβ3 activation in response to convulxin, however, platelets activated with DxS under the same conditions retained the ability to activate αIIbβ3. In response to DxS, platelet aggregation, intracellular calcium mobilization, and αIIbβ3 activation were significantly inhibited in platelets pre-treated with PI-3K inhibitors. Taken together these results for the first time establish a novel tyrosine kinase pathway in platelets that cause fibrinogen receptor activation in a PI-3K dependent manner without a role for Syk. In conclusion, we have evaluated the role of myosin light chain kinase, Syk, and PI-3 kinase downstream of platelet receptor-mediated pathways. We have examined the phosphorylation status of several of their effector molecules and have correlated these events with their functional responses in platelets. Here we have highlighted several roles for platelet kinases and their relative importance in regulating platelet functional outcomes. / Physiology

Thesis advisor: Maitreyi E. Das / Polarization allows cells to form and maintain morphologies necessary for their diverse functions during processes such as growth, division, differentiation, and migration. Signaling proteins such as the family of small Rho GTPases promote polarization by spatiotemporally regulating cytoskeleton dynamics and coordinating membrane trafficking. Here, we investigate and define roles of the Rho GTPase Cdc42 in promoting polarization in S. pombe. As fission yeast, S. pombe cells grow from their cell ends during interphase and divide by medial fission to produce two new daughter cells. As cell-walled organisms, growth and division require intricate remodeling and expansion of the cell wall via incorporation of new membrane and proteins at these polarized sites. Thus, growth and division require specific sequences of membrane trafficking events to deliver and remove cargo at appropriate times and locations. During cytokinesis, fission yeast cells divide by synthesizing new cell wall called the septum to medially bisect the cell. The septum is synthesized behind the actomyosin ring to aid its constriction. Once ring constriction completes and the septum matures, the septum is partially digested to physically separate the daughter cells. Previous work has shown that Cdc42 promotes the delivery of specific but not all septum-synthesizing enzymes as well as septum-digesting enzymes, but it was not known how Cdc42 activation is regulated at the division site to temporally coordinate these processes. Here, we show that the Cdc42 GAPs Rga4 and Rga6 promote proper septum synthesis and timely cell separation by locally decreasing Cdc42 activation during late cytokinesis. This work also reveals a role for Cdc42 in regulating clathrin-mediated endocytosis, both at the division site as well as at growing cell ends. To further explore this role, we systematically examined the behaviors of endocytic actin patches in mutants of Cdc42 regulators and compared these dynamics to wild-type controls. This characterization led to the observation that endocytic patches are best formed to induce successful patch internalization at sites of polarization where Cdc42 is active. In this work, we show that Cdc42 activation promotes proper endocytic patch behavior in a dose-dependent manner and that Cdc42 regulates endocytosis via its downstream effector, the Pak1 kinase. We also demonstrate that Cdc42 and Pak1 activity promote endocytosis through at least two pathways which regulate branched actin formation. First, we show that Cdc42 and Pak1 promote proper endocytic actin patch formation. Secondly, we show that Pak1-mediated phosphorylation of the endocytic Type I myosin promotes timely internalization of endocytic actin patches. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

A novel luminescence resonance energy transfer (LRET) nanocircuit assay involving a donor and two acceptors in tandem was developed to study the dynamic interaction of skeletal muscle contraction proteins. The donor transmits energy relayed to the acceptors distinguishing myosin subfragment-1 (S1) lever arm orientations. The last acceptor allows the detection of S1's bound near or in between troponin complexes on the thin filament. Additionally, calcium related changes between troponin T and myosin were detected. Based on this data, the troponin complex situated every 7 actin monomers, hinders adjacently bound myosins to complete their power stroke; whereas myosins bound in between troponin complexes undergo complete power strokes.

Familial Hypertrophic cardiomyopathy (HCM) causes ventricle walls to thicken and often leads to sudden death especially in adults. Mutations in the subfragment 2 (S2) of β-cardiac myosin are implicated in the genetic disorder. This S2 region is a coiled-coil rod region resulting from the dimeric form of myosin II. It has been proposed that an elastic quality allows normal S2 to absorb force during the powerstroke according to the sliding filament model. To test the flexibility of single molecules of S2 against levels of physiological force, the Gravitational Force Spectrometer (GFS) is being developed. This novel system employs a standard microscope on an equatorial mount that allows the spectrometer to be rotated freely in space. Stationary glass beads are attached to a microscope slide where the molecule is tethered between the stationary bead and a smaller mobile bead. The GFS is oriented so that the force of gravity can act on the mobile bead and so impart a small force to the tethered subfragment. Additionally, a video system in conjunction with ImageJ software makes a distance measurement of the molecule possible with a resolution of around 11 nm. The S2 can be stretched parallel or perpendicular to the coiled coil to elucidate different structural properties of the rod. This study is the first to show structural evidence that S2 in vertebrate skeletal myosin uncoils proportionally to physiological force loads. Because of this, the usefulness and promise of the novel GFS is highlighted, and the biological role of S2's flexibility can be directly commented on. If the dimer undergoes uncoiling at physiological force loads as shown, then it is reasonable to think that this might occur in nature in response to the stress of the powerstroke on a single molecule. This unwinding could be to absorb force as a mechanism to protect the muscle fiber.

Mechanistic details on the regulation of striated muscle contraction still need to be determined, particularly the specific structural locations of the elements comprising the thick and thin filaments. Of special interest is the location of the regulatory component, troponin, on the actin filament and how its presence influences the behavior of myosin binding to the thin filament. In the present study: (1) Luminescence resonance energy transfer was used to monitor potential conformational changes in the reconstituted thin filament between the C-terminal region of troponin T and myosin subfragment 1; (2) Location of troponin in previously derived atomic models of the acto-myosin complex was mapped to visualize specific contacts; and (3) Shortened tropomyosin was engineered and protein binding and ATPase assays were performed to study the effect of myosin binding close to the troponin complex. Analysis of the results suggest the following: (1) Irrespective of calcium levels, the C-terminal region of troponin T is located close to myosin loop 3 and a few actin helices that may perturb strong acto-myosin interactions responsible for force production. (2) Atomic models indicate myosin subfragment 1 cannot attain the post- powerstroke state due to the full motion of the lever arm being sterically hindered by troponin. (3) A shortened tropomyosin with five actin binding modules (instead of the native seven in muscle cells) binds actin contiguously in a head-to-tail manner and serves to increase the periodicity of troponin complexes on the actin filament. Such behavior eliminates the structure of the actin filament being responsible for the binding location of tropomyosin. (4) Differential behavior of myosin subfragment 1 i.e. (a) binding adjacent to troponin and (b) binding further away from troponin, is apparent as tropomyosin and troponin appear to govern the regions or "target zones" where myosin can bind productively along the actin filament. Physiologically, myosins able to bind close to troponin, but not participate in force production may function as mechanical sensors to attenuate or dampen the force generated from the so-called "target zones". Therefore, this could be a pseudo-regulatory mechanism that functions to protect the contractile apparatus from damage.

Hypertrophic cardiomyopathy (HCM), a heart-related abnormality, is the most prevalent cause of sudden death in young athletes at sporting events. A cluster of cardiomyopathy mutations are localized in β-cardiac myosin at the N-terminal region of subfragment-2. Using resonance energy transfer probes, a synthetic peptide model system was developed to study stability of the coiled coil (S2 fragment) structure by determining monomer-dimer equilibrium of the peptide. Fluorescence resonance energy transfer and MacroModel software suite were used to obtain distance measurements along with measurement of coiled coil formation. The model peptide was used to characterize the effects of disease-causing-mutations and examine potential candidate drugs (polyamines) to counteract effects of mutations causing HCM. Distance measurements between donor and acceptor probes obtained by computational simulation and fluorescence resonance energy transfer (FRET) were consistent. Measurements also agreed with simulations of unlabeled wildtype, indicating coiled coil structural stability of the peptide. Interaction of the site-specific antibody with the peptide strongly inhibited dimerization and destabilized coiled coil structure of the peptide. Presence of negatively charged glutamate residues in the region of subfragment-2 strongly suggested a potential interaction site for positively charged polyamines. Binding of certain polyamines, such as poly-L-Lysine 11 residues and poly-D-Lysine 17 residues, demonstrated the ability to enhance dimerization and improve stability of the coiled coil structure, while some other polyamines were shown to have insignificant impact on the structure. In an attempt to characterize the effect of HCM-causing-mutations, peptides containing E924K mutation and lethal mutation E930 deletion were synthesized. Fluorescence resonance probes were conjugated to the mutant peptides to determine coiled coil formation. Results obtained from both dynamic simulations and resonance energy transfer experiments indicated that these mutations strongly inhibit dimerization, and thus, destabilize coiled coil structure of the peptide. Further experiments were conducted using heterodimers containing a chain of wildtype and a chain of mutant peptide. Both E924K & Edel930 mutations destabilized coiled coil formation and prevented dimerization. This peptide model system would provide a promising tool for drug development targeting HCM-causing-mutations along the S2 region of myosin.