It has been shown that reactive oxygen species (ROS) are released in response to articular cartilage injury. The excessive release of ROS has been shown to be mitochondrial in nature and leads to chondrocyte death which in turn can lead to post-traumatic osteoarthritis (PTOA). Evidence suggests that mitochondria are attached to chondrocytes' cytoskeleton. Upon tissue level deformation, it is believed that mitochondria also experience deformation in response to cytoskeletal strain, releasing ROS. Therefore, it was hypothesized that inhibition of chondrocytes' cytoskeleton would prevent mitochondrial distortion rendering them unable to release ROS in response to the applied strain, saving chondrocytes. Osteochondral explants treated with cytoskeletal inhibitors were found to reduce mitochondrial ROS production directly after impact and increase chondrocyte viability 24 hours after impact. The release of mitochondrial ROS is an important mechanotranduction pathway in the initiation of PTOA.

It is becoming clear that mechanical stimuli are critical in regulating cell biology; however, the short-term structural response of a cell to mechanical forces remains relatively poorly understood. We mechanically stimulated cells expressing actin-EGFP with controlled forces (0-20nN) in order to investigate the cell’s structural response. Two clear force dependent responses were observed: a short-term local deformation of actin stress fibres and a long-term force-induced remodelling of stress fibres at cell edges, far from the point of contact. We were also able to quantify strain dynamics occurring along stress fibres. The cell exhibits complex heterogeneous negative and positive strain fluctuations along stress fibres, indicating localized dynamic contraction and expansion. A ~50% increase in myosin contractile activity is apparent following application of 20nN force. Directly visualizing force-propagation and stress fibre strain dynamics has revealed new information about the pathways involved in mechanotransduction which ultimately govern the downstream response of a cell.

Filamentous fungi, such as <i>Aspergillus nidulans</i>, are composed of tubular, highly polarized, multinucleate cells called hyphae. Polar growth involves secretion specifically at the hyphal tip. Secretion involves intracellular transport and co-ordination of the cytoskeleton and the endomembrane system. <p>Intracellular transport is likely mediated by cytoskeletal elements, which, in fungal cells consist primarily of actin and microtubules (MTs). An <i>A. nidulans</i> strain transformed with green fluorescent protein (GFP) tagged α-tubulin was utilized in the investigation of relationship between cytoplasmic MT arrays and hyphal growth rate. <i>A. nidulans</i> MTs were observed to be long and flexuous and to run roughly parallel to the long axis of hyphae. No correlation between relative MT abundance and hyphal growth rate was observed, although non-growing hyphae had a lower relative MT abundance than growing hyphae. Actin depolymerization decreased hyphal growth rate while MT depolymerization did not. MT stabilization increased hyphal growth rate. Ethanol, the solvent in which the MT and actin inhibitors were dissolved, increased both average overall growth rate and growth rate variability for individual hyphae. Taxol appeared to interact with irradiation to decreased growth rate during imaging. <p>Golgi are involved in secretion and potentially in polar growth. An <i>A. nidulans</i> α-coatomer protein (COP)I homolog (α-COPI), tagged with GFP, was used to investigate the role(s) of fungal Golgi in polar growth. α-COPI-GFP co-localized with the known Golgi marker, α-2,6-sialyltransferase (ST), tagged with red fluorescent protein (RFP), in untreated hyphae. Based on this observation, I propose that α-COPI-GFP can be used as a proxy for fungal Golgi localization. Fungal Golgi were more abundant at hyphal tips than subapically. Fungal Golgi forward (tipward) velocity correlated with hyphal growth rate. Fungal Golgi forward velocity was, on average, approximately ten times greater than average hyphal growth rate. Actin depolymerization reduced fungal Golgi forward velocity while MT depolymerization did not. However, MT stabilization increased fungal Golgi forward velocity. <p>Polymerized MTs do not appear to be essential for hyphal growth but do appear to be involved in hyphal growth rate variability. MTs also appear to play some role in the movement of fungal Golgi. The distribution and movement of fungal Golgi is clearly related to polarity.

Transient Receptor Potential ‘Melastatin’ 7 (TRPM7) is a ubiquitously expressed, non-selective divalent cation channel implicated in diverse cellular functions including actomyosin cytoskeletal remodeling, magnesium homeostasis, and anoxic neuronal death. The present study investigates the role of TRPM7 in modulating neuronal morphology and regulating neuronal cytoskeletal dynamics after anoxia. Overexpression of GFP-tagged TRPM7 in neuronal cultures caused a stunted morphology with fewer neurite branches than controls, suggesting that TRPM7 regulates the neuronal cytoskeleton during dendritic outgrowth. I have discovered that TRPM7 may regulate morphology via activation of cofilin-1 (an actin binding protein). I found that TRPM7-dependent cofilin activation during anoxia mediated neuronal death. Overall my work reveals a novel link between anoxia-induced TRPM7 activity and cofilin activation, which likely contributes to neurodegeneration after ischemia.

Transient Receptor Potential ‘Melastatin’ 7 (TRPM7) is a ubiquitously expressed, non-selective divalent cation channel implicated in diverse cellular functions including actomyosin cytoskeletal remodeling, magnesium homeostasis, and anoxic neuronal death. The present study investigates the role of TRPM7 in modulating neuronal morphology and regulating neuronal cytoskeletal dynamics after anoxia. Overexpression of GFP-tagged TRPM7 in neuronal cultures caused a stunted morphology with fewer neurite branches than controls, suggesting that TRPM7 regulates the neuronal cytoskeleton during dendritic outgrowth. I have discovered that TRPM7 may regulate morphology via activation of cofilin-1 (an actin binding protein). I found that TRPM7-dependent cofilin activation during anoxia mediated neuronal death. Overall my work reveals a novel link between anoxia-induced TRPM7 activity and cofilin activation, which likely contributes to neurodegeneration after ischemia.

It is becoming clear that mechanical stimuli are critical in regulating cell biology; however, the short-term structural response of a cell to mechanical forces remains relatively poorly understood. We mechanically stimulated cells expressing actin-EGFP with controlled forces (0-20nN) in order to investigate the cell’s structural response. Two clear force dependent responses were observed: a short-term local deformation of actin stress fibres and a long-term force-induced remodelling of stress fibres at cell edges, far from the point of contact. We were also able to quantify strain dynamics occurring along stress fibres. The cell exhibits complex heterogeneous negative and positive strain fluctuations along stress fibres, indicating localized dynamic contraction and expansion. A ~50% increase in myosin contractile activity is apparent following application of 20nN force. Directly visualizing force-propagation and stress fibre strain dynamics has revealed new information about the pathways involved in mechanotransduction which ultimately govern the downstream response of a cell.

The teratogenic drugs cytochalasin B and valproic acid have been shown to alter F-actin polymerization, an effect that is crucial in forming microfilaments. Microfilaments form important cytoskeletal structures that maintain the structural integrity of the cell, cause cell motility and cell migration. Microfilament alterations are known to cause neural tube defects such as spina bifida and anencephaly (Walmod et al., 1999). We here aim to show that disruption of microfilaments by cytochalasin B and valproic acid affects the tensile properties of the tissue. Biomechanics is an interdisciplinary field that allows mechanical concepts to help us understand embryo development. This project used a novel tissue stretching device that measures the tensile properties of neural and epidermal tissue. The instrument used a pair of cantilevered wires to which the specimen was glued. This device stretched the mid-neural and -lateral tissue anterior-posterior (AP) and medio-lateral (ML) unidirectionally. The tensile properties of the tissue were determined by Resultant Young’s Modulus that depends on the true stress and true strain in the tissue sample. The experiment was conducted at a strain rate of 50%. Axolotl embryos were treated with 5ug/mL and 2.5ug/mL cytochalasin B and 5mM valproic acid at stage 13 (early neurula) for an hour, washed, and allowed to develop to stage 15 before it was used in the uniaxial tissue stretcher. Changes in the F-actin filaments were analysed by phalloidin staining and viewed under a confocal microscope. The tests show that disruption of microfilaments by cytochalasin B increases the stiffness of the dorsal-tissue by as much as 101% for CB-treated tissues stretched in the AP direction and 298% when stretched in the ML direction. VA-treated neural plate tissue showed a stiffness increase of 278% when stretched in the AP direction and 319%, when stretched in the ML direction. Changes in the F-actin filaments are quantified by phalloidin staining viewed with confocal microscopy. These findings indicate that direction-dependent mechanical forces in the tissue are contributing factors in closure of the neural tube in axolotl embryos.

The cytoskeleton plays an important role in trafficking proteins and other macromolecular moieties throughout the cell. Viruses have been thought to depend heavily on the cytoskeleton for their replication cycles. However, studies, including one in our lab, found that some viruses are not inhibited by anti-microtubule drugs. This study was undertaken to evaluate the replication of viruses from several families in the presence of cytoskeleton-inhibiting drugs and to examine the intracellular localization of the proteins of one of these viruses, Sindbis virus, to test the hypothesis that alternate pathways are used if the cytoskeleton is inhibited. We found that Sindbis virus (Togaviridae, positive-strand RNA), vesicular stomatitis virus (Rhabdoviridae, negative-strand RNA), and Herpes simplex virus 1 (Herpesviridae, DNA virus) were not inhibited by these drugs, contrary to expectation. Differences in the localization of the Sindbis virus were observed, suggesting the existence of alternate pathways for intracellular transport.

The teratogenic drugs cytochalasin B and valproic acid have been shown to alter F-actin polymerization, an effect that is crucial in forming microfilaments. Microfilaments form important cytoskeletal structures that maintain the structural integrity of the cell, cause cell motility and cell migration. Microfilament alterations are known to cause neural tube defects such as spina bifida and anencephaly (Walmod et al., 1999). We here aim to show that disruption of microfilaments by cytochalasin B and valproic acid affects the tensile properties of the tissue. Biomechanics is an interdisciplinary field that allows mechanical concepts to help us understand embryo development. This project used a novel tissue stretching device that measures the tensile properties of neural and epidermal tissue. The instrument used a pair of cantilevered wires to which the specimen was glued. This device stretched the mid-neural and -lateral tissue anterior-posterior (AP) and medio-lateral (ML) unidirectionally. The tensile properties of the tissue were determined by Resultant Young’s Modulus that depends on the true stress and true strain in the tissue sample. The experiment was conducted at a strain rate of 50%. Axolotl embryos were treated with 5ug/mL and 2.5ug/mL cytochalasin B and 5mM valproic acid at stage 13 (early neurula) for an hour, washed, and allowed to develop to stage 15 before it was used in the uniaxial tissue stretcher. Changes in the F-actin filaments were analysed by phalloidin staining and viewed under a confocal microscope. The tests show that disruption of microfilaments by cytochalasin B increases the stiffness of the dorsal-tissue by as much as 101% for CB-treated tissues stretched in the AP direction and 298% when stretched in the ML direction. VA-treated neural plate tissue showed a stiffness increase of 278% when stretched in the AP direction and 319%, when stretched in the ML direction. Changes in the F-actin filaments are quantified by phalloidin staining viewed with confocal microscopy. These findings indicate that direction-dependent mechanical forces in the tissue are contributing factors in closure of the neural tube in axolotl embryos.

Filamentous fungi, such as <i>Aspergillus nidulans</i>, are composed of tubular, highly polarized, multinucleate cells called hyphae. Polar growth involves secretion specifically at the hyphal tip. Secretion involves intracellular transport and co-ordination of the cytoskeleton and the endomembrane system. <p>Intracellular transport is likely mediated by cytoskeletal elements, which, in fungal cells consist primarily of actin and microtubules (MTs). An <i>A. nidulans</i> strain transformed with green fluorescent protein (GFP) tagged α-tubulin was utilized in the investigation of relationship between cytoplasmic MT arrays and hyphal growth rate. <i>A. nidulans</i> MTs were observed to be long and flexuous and to run roughly parallel to the long axis of hyphae. No correlation between relative MT abundance and hyphal growth rate was observed, although non-growing hyphae had a lower relative MT abundance than growing hyphae. Actin depolymerization decreased hyphal growth rate while MT depolymerization did not. MT stabilization increased hyphal growth rate. Ethanol, the solvent in which the MT and actin inhibitors were dissolved, increased both average overall growth rate and growth rate variability for individual hyphae. Taxol appeared to interact with irradiation to decreased growth rate during imaging. <p>Golgi are involved in secretion and potentially in polar growth. An <i>A. nidulans</i> α-coatomer protein (COP)I homolog (α-COPI), tagged with GFP, was used to investigate the role(s) of fungal Golgi in polar growth. α-COPI-GFP co-localized with the known Golgi marker, α-2,6-sialyltransferase (ST), tagged with red fluorescent protein (RFP), in untreated hyphae. Based on this observation, I propose that α-COPI-GFP can be used as a proxy for fungal Golgi localization. Fungal Golgi were more abundant at hyphal tips than subapically. Fungal Golgi forward (tipward) velocity correlated with hyphal growth rate. Fungal Golgi forward velocity was, on average, approximately ten times greater than average hyphal growth rate. Actin depolymerization reduced fungal Golgi forward velocity while MT depolymerization did not. However, MT stabilization increased fungal Golgi forward velocity. <p>Polymerized MTs do not appear to be essential for hyphal growth but do appear to be involved in hyphal growth rate variability. MTs also appear to play some role in the movement of fungal Golgi. The distribution and movement of fungal Golgi is clearly related to polarity.