The plant cell wall, plasma membrane and cytoskeleton exist as a cell surface continuum. This int... more The plant cell wall, plasma membrane and cytoskeleton exist as a cell surface continuum. This interconnection of organelles forms the interface between the plant cell and the external environment and is important for detecting the presence of a diverse range of stimuli. A plethora of plasma membrane microdomains with putative roles in membrane localized enzymatic or signalling processes have been described. While regulation of cell wall composition is defined by proteins within the plasma membrane, the cell wall has been shown to have an anchoring role on plasma membrane proteins which affects their lateral mobility. This interplay between plasma membrane and cell wall components is necessary for plasma membrane microdomain function. Actin and microtubule cytoskeletons are also involved in maintenance and function of the cell surface continuum. Investigation of the interactions between organellar components of this mechanism are important if we are to understand how cells respond to...
Studying protein diffusion informs us about how proteins interact with their environment. Work on... more Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.
Methods in molecular biology (Clifton, N.J.), 2013
Developmental biologists require methods for marking cell lineages as they arise in living tissue... more Developmental biologists require methods for marking cell lineages as they arise in living tissues. Traditionally, lineages have been traced in fixed tissues but these observations are difficult to verify. We present a method by which a progenitor cell and all of its lineage become marked by a nuclear-localised fluorescent protein. This allows rapid estimation of the effects of genetic or physical manipulation of developing tissues. Heat shock is used to activate YFP expression in single progenitor cells which is heritable by all daughter cells in subsequent rounds of mitosis. Heat shock can be applied to specimens generally using an incubator to generate random lineage patterns or more specifically to single cells or small regions using laser activation of the lineage marking system.
Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of ba... more Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of basic concepts in cell biology. The protocol presented here has underpinned much of the in vivo results highlighting the dynamic nature of the plant secretory pathway. Transient transformation of tobacco leaf epidermal cells is a relatively fast technique to assess expression of genes of interest. These cells can be used to generate stable plant lines using a more time-consuming, cell culture technique. Transient expression takes from 2 to 4 days whereas stable lines are generated after approximately 2 to 4 months.
Components of the plant cell secretory pathway, including the endoplasmic reticulum and Golgi app... more Components of the plant cell secretory pathway, including the endoplasmic reticulum and Golgi apparatus, are in constant motion. The photoactivation of GFP has been used to determine that proteins within the membrane of the ER flow as the ER is remodelled. Measurement of the rate at which activated GFP moves away from the activation spot shows that this motion is much faster than would be expected if membrane components moved simply by diffusion. Treatment with latrunculin to depolymerize the actin cytoskeleton stops ER remodelling and reduces the rate of GFP movement to that expected from diffusion alone. This suggests that myosin binds directly or indirectly to ER membrane proteins and actively moves them around over the actin scaffold. Tracking of Golgi body movement was used to demonstrate that they move at the same rate and in the same direction as do photoactivated ER surface proteins. Golgi bodies, therefore, move with, and not over, the surface of the ER. These observations ...
Proceedings of the National Academy of Sciences of the United States of America, Jan 25, 2006
A challenging task in the study of the secretory pathway is the identification and localization o... more A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments. With this approach, the density gradient distributions of 689 proteins from Arabidopsis thaliana were determined, enabling confident and simultaneous localization of 527 proteins to the endoplasmic reticulum, Golgi apparatus, vacuolar membrane, plasma membrane, or mitochondria and plastids. This parallel analysis of endomembrane components has enabled protein steady-state distributi...
The genetic control of plant development is mediated by cellular interactions, and we have develo... more The genetic control of plant development is mediated by cellular interactions, and we have developed new optical and genetic techniques for manipulating the fates of cells inside living mer-istems. The Arabidopsis root meristem provides an ideal model system for investigating ...
... AUTHORS Imogen A. Sparkes, John Runions, Anne Kearns, Chris Hawes ... 250mg D-glucose, 5ml ME... more ... AUTHORS Imogen A. Sparkes, John Runions, Anne Kearns, Chris Hawes ... 250mg D-glucose, 5ml MES stock solution, 5ml Na3PO4.12H2O stock solution, 5µl 1 M acetosyringone stock solution, make up to 50ml with dH2O. Infiltration medium stock solutions: ...
Controlled movement of the nucleus is important in a wide variety of plant cellular events. Posit... more Controlled movement of the nucleus is important in a wide variety of plant cellular events. Positioning involving intact nuclei occurs in cell division, development, tip growing systems such as the root hair and in response to stimuli, including light, touch and infection. Positioning is also essential in the division and replication of nuclear components, ranging from chromosome attachment to the breakdown and reformation of the nuclear envelope. Although description and understanding of the processes involved have advanced rapidly in recent years, significant gaps remain in our knowledge, especially concerning nuclear proteins involved in anchoring and interacting with cytoskeletal and nucleoskeletal elements involved in movement. In the present review, processes involving the movement and positioning of nuclei and nuclear components are described together with novel proteins implicated in nucleoskeletal and cytoskeletal interactions.
Protein localization in living plant cells is commonly studied using fluorescent protein fusions.... more Protein localization in living plant cells is commonly studied using fluorescent protein fusions. Stable transformation of plant cells requires the use of binary vectors, which are larger and not as amenable to genetic manipulation as animal cell transfection vectors. Binary vectors containing fluorescent protein fusion constructs are prepared using standard molecular biological techniques. Fusion genes as well as promoters and selection markers are stably incorporated into the plant cell genome via Agrobacterium-mediated transfer. Presented here are a series of protocols that detail binary vector construction, bacterial transformation, and a rapid transient assay technique that can be used to evaluate fusion protein fluorescence in leaves.
The plant cell wall, plasma membrane and cytoskeleton exist as a cell surface continuum. This int... more The plant cell wall, plasma membrane and cytoskeleton exist as a cell surface continuum. This interconnection of organelles forms the interface between the plant cell and the external environment and is important for detecting the presence of a diverse range of stimuli. A plethora of plasma membrane microdomains with putative roles in membrane localized enzymatic or signalling processes have been described. While regulation of cell wall composition is defined by proteins within the plasma membrane, the cell wall has been shown to have an anchoring role on plasma membrane proteins which affects their lateral mobility. This interplay between plasma membrane and cell wall components is necessary for plasma membrane microdomain function. Actin and microtubule cytoskeletons are also involved in maintenance and function of the cell surface continuum. Investigation of the interactions between organellar components of this mechanism are important if we are to understand how cells respond to...
Studying protein diffusion informs us about how proteins interact with their environment. Work on... more Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.
Methods in molecular biology (Clifton, N.J.), 2013
Developmental biologists require methods for marking cell lineages as they arise in living tissue... more Developmental biologists require methods for marking cell lineages as they arise in living tissues. Traditionally, lineages have been traced in fixed tissues but these observations are difficult to verify. We present a method by which a progenitor cell and all of its lineage become marked by a nuclear-localised fluorescent protein. This allows rapid estimation of the effects of genetic or physical manipulation of developing tissues. Heat shock is used to activate YFP expression in single progenitor cells which is heritable by all daughter cells in subsequent rounds of mitosis. Heat shock can be applied to specimens generally using an incubator to generate random lineage patterns or more specifically to single cells or small regions using laser activation of the lineage marking system.
Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of ba... more Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of basic concepts in cell biology. The protocol presented here has underpinned much of the in vivo results highlighting the dynamic nature of the plant secretory pathway. Transient transformation of tobacco leaf epidermal cells is a relatively fast technique to assess expression of genes of interest. These cells can be used to generate stable plant lines using a more time-consuming, cell culture technique. Transient expression takes from 2 to 4 days whereas stable lines are generated after approximately 2 to 4 months.
Components of the plant cell secretory pathway, including the endoplasmic reticulum and Golgi app... more Components of the plant cell secretory pathway, including the endoplasmic reticulum and Golgi apparatus, are in constant motion. The photoactivation of GFP has been used to determine that proteins within the membrane of the ER flow as the ER is remodelled. Measurement of the rate at which activated GFP moves away from the activation spot shows that this motion is much faster than would be expected if membrane components moved simply by diffusion. Treatment with latrunculin to depolymerize the actin cytoskeleton stops ER remodelling and reduces the rate of GFP movement to that expected from diffusion alone. This suggests that myosin binds directly or indirectly to ER membrane proteins and actively moves them around over the actin scaffold. Tracking of Golgi body movement was used to demonstrate that they move at the same rate and in the same direction as do photoactivated ER surface proteins. Golgi bodies, therefore, move with, and not over, the surface of the ER. These observations ...
Proceedings of the National Academy of Sciences of the United States of America, Jan 25, 2006
A challenging task in the study of the secretory pathway is the identification and localization o... more A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments. With this approach, the density gradient distributions of 689 proteins from Arabidopsis thaliana were determined, enabling confident and simultaneous localization of 527 proteins to the endoplasmic reticulum, Golgi apparatus, vacuolar membrane, plasma membrane, or mitochondria and plastids. This parallel analysis of endomembrane components has enabled protein steady-state distributi...
The genetic control of plant development is mediated by cellular interactions, and we have develo... more The genetic control of plant development is mediated by cellular interactions, and we have developed new optical and genetic techniques for manipulating the fates of cells inside living mer-istems. The Arabidopsis root meristem provides an ideal model system for investigating ...
... AUTHORS Imogen A. Sparkes, John Runions, Anne Kearns, Chris Hawes ... 250mg D-glucose, 5ml ME... more ... AUTHORS Imogen A. Sparkes, John Runions, Anne Kearns, Chris Hawes ... 250mg D-glucose, 5ml MES stock solution, 5ml Na3PO4.12H2O stock solution, 5µl 1 M acetosyringone stock solution, make up to 50ml with dH2O. Infiltration medium stock solutions: ...
Controlled movement of the nucleus is important in a wide variety of plant cellular events. Posit... more Controlled movement of the nucleus is important in a wide variety of plant cellular events. Positioning involving intact nuclei occurs in cell division, development, tip growing systems such as the root hair and in response to stimuli, including light, touch and infection. Positioning is also essential in the division and replication of nuclear components, ranging from chromosome attachment to the breakdown and reformation of the nuclear envelope. Although description and understanding of the processes involved have advanced rapidly in recent years, significant gaps remain in our knowledge, especially concerning nuclear proteins involved in anchoring and interacting with cytoskeletal and nucleoskeletal elements involved in movement. In the present review, processes involving the movement and positioning of nuclei and nuclear components are described together with novel proteins implicated in nucleoskeletal and cytoskeletal interactions.
Protein localization in living plant cells is commonly studied using fluorescent protein fusions.... more Protein localization in living plant cells is commonly studied using fluorescent protein fusions. Stable transformation of plant cells requires the use of binary vectors, which are larger and not as amenable to genetic manipulation as animal cell transfection vectors. Binary vectors containing fluorescent protein fusion constructs are prepared using standard molecular biological techniques. Fusion genes as well as promoters and selection markers are stably incorporated into the plant cell genome via Agrobacterium-mediated transfer. Presented here are a series of protocols that detail binary vector construction, bacterial transformation, and a rapid transient assay technique that can be used to evaluate fusion protein fluorescence in leaves.
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