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A systematic analysis of the PARP protein family identifies new functions critical for cell physiology.

Vyas S, Chesarone-Cataldo M, Todorova T, Huang YH, Chang P - Nat Commun (2013)

Bottom Line: These include the regulation of membrane structures, cell viability, cell division and the actin cytoskeleton.Further analysis of PARP14 shows that it is a component of focal adhesion complexes required for proper cell motility and focal adhesion function.In total, we show that PARP proteins are critical regulators of eukaryotic physiology.

View Article: PubMed Central - PubMed

Affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

ABSTRACT
The poly(ADP-ribose) polymerase (PARP) family of proteins use NAD(+) as their substrate to modify acceptor proteins with ADP-ribose modifications. The function of most PARPs under physiological conditions is unknown. Here, to better understand this protein family, we systematically analyse the cell cycle localization of each PARP and of poly(ADP-ribose), a product of PARP activity, then identify the knockdown phenotype of each protein and perform secondary assays to elucidate function. We show that most PARPs are cytoplasmic, identify cell cycle differences in the ratio of nuclear to cytoplasmic poly(ADP-ribose) and identify four phenotypic classes of PARP function. These include the regulation of membrane structures, cell viability, cell division and the actin cytoskeleton. Further analysis of PARP14 shows that it is a component of focal adhesion complexes required for proper cell motility and focal adhesion function. In total, we show that PARP proteins are critical regulators of eukaryotic physiology.

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PARP14 depletion from focal adhesions results in increased adhesive strengthA) Control or PARP14 siRNA treated cells plated on fibronectin were treated with trypsin for the indicated times and percent cells detached quantified at each time point. Error bars represent standard deviation. B) Control or PARP14 siRNA treated cells plated on fibronectin were subjected to a constant centrifugal force (2,000 g) for 30 min and the number of cells detached during centrifugation quantified. Representative images of control and PARP14 knock-down cells before and after centrifugation shown at right. Error bars represent standard deviation. C) Control or PARP14 siRNA treated cells were allowed to adhere to a fibronectin coated plate for the indicated times, fixed and stained with anti-paxillin and phalloidin, and the area of individual cells quantified at each timepoint. Error bars represent standard deviation. Representative images of control or PARP14 knock-down cells at each time point are shown. Scale bar, 25 μm.
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Figure 6: PARP14 depletion from focal adhesions results in increased adhesive strengthA) Control or PARP14 siRNA treated cells plated on fibronectin were treated with trypsin for the indicated times and percent cells detached quantified at each time point. Error bars represent standard deviation. B) Control or PARP14 siRNA treated cells plated on fibronectin were subjected to a constant centrifugal force (2,000 g) for 30 min and the number of cells detached during centrifugation quantified. Representative images of control and PARP14 knock-down cells before and after centrifugation shown at right. Error bars represent standard deviation. C) Control or PARP14 siRNA treated cells were allowed to adhere to a fibronectin coated plate for the indicated times, fixed and stained with anti-paxillin and phalloidin, and the area of individual cells quantified at each timepoint. Error bars represent standard deviation. Representative images of control or PARP14 knock-down cells at each time point are shown. Scale bar, 25 μm.

Mentions: For cells to properly retract membrane protrusions, they must be able to efficiently breakdown FAs in a process called FA turnover47. The inability of PARP14 knock-down cells to properly retract membrane protrusions, combined with its localization to FAs, suggested that PARP14 functions in FA turnover. Therefore we examined FA adhesive force in control and PARP14 knock-downs using three independent assays. First we utilized a trypsin-based detachment assay measuring time required to detach cells from substrate48. PARP14 knockdown cells were more resistant to trypsinization than controls, with ~ 2 times more cells remaining adhered after similar periods of trypsinization (Figure 6A). Second we employed a centrifugation assay comparing the number of cells remaining adhered to substrate after constant application of centrifugal force. Approximately 5 times more PARP14 knock-down cells remained adhered to substrate after centrifugation compared to controls (Figure 6B). Third, we used a cell-spreading assay measuring the area of cell spreading per unit time (Figure 6C). At early time points when cells adhered to substrate but had not yet formed defined FAs, control and PARP14 knock-downs were of similar size suggesting that the morphological defects observed in PARP14 knock-downs were dependent on FA formation and not simply a property of the cells themselves. As cells assembled FAs and spread onto substrate, PARP14 knock-down cells covered ~twice the surface area of control cells at each time point (Figure 6C). These three independent assays suggest that the adhesive strength between FAs and substrate is stronger in PARP14 knock-downs relative to control. It is likely that this increased adhesive strength leads to slowed FA disassembly and prevents cellular protrusions from properly retracting.


A systematic analysis of the PARP protein family identifies new functions critical for cell physiology.

Vyas S, Chesarone-Cataldo M, Todorova T, Huang YH, Chang P - Nat Commun (2013)

PARP14 depletion from focal adhesions results in increased adhesive strengthA) Control or PARP14 siRNA treated cells plated on fibronectin were treated with trypsin for the indicated times and percent cells detached quantified at each time point. Error bars represent standard deviation. B) Control or PARP14 siRNA treated cells plated on fibronectin were subjected to a constant centrifugal force (2,000 g) for 30 min and the number of cells detached during centrifugation quantified. Representative images of control and PARP14 knock-down cells before and after centrifugation shown at right. Error bars represent standard deviation. C) Control or PARP14 siRNA treated cells were allowed to adhere to a fibronectin coated plate for the indicated times, fixed and stained with anti-paxillin and phalloidin, and the area of individual cells quantified at each timepoint. Error bars represent standard deviation. Representative images of control or PARP14 knock-down cells at each time point are shown. Scale bar, 25 μm.
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Related In: Results  -  Collection

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Figure 6: PARP14 depletion from focal adhesions results in increased adhesive strengthA) Control or PARP14 siRNA treated cells plated on fibronectin were treated with trypsin for the indicated times and percent cells detached quantified at each time point. Error bars represent standard deviation. B) Control or PARP14 siRNA treated cells plated on fibronectin were subjected to a constant centrifugal force (2,000 g) for 30 min and the number of cells detached during centrifugation quantified. Representative images of control and PARP14 knock-down cells before and after centrifugation shown at right. Error bars represent standard deviation. C) Control or PARP14 siRNA treated cells were allowed to adhere to a fibronectin coated plate for the indicated times, fixed and stained with anti-paxillin and phalloidin, and the area of individual cells quantified at each timepoint. Error bars represent standard deviation. Representative images of control or PARP14 knock-down cells at each time point are shown. Scale bar, 25 μm.
Mentions: For cells to properly retract membrane protrusions, they must be able to efficiently breakdown FAs in a process called FA turnover47. The inability of PARP14 knock-down cells to properly retract membrane protrusions, combined with its localization to FAs, suggested that PARP14 functions in FA turnover. Therefore we examined FA adhesive force in control and PARP14 knock-downs using three independent assays. First we utilized a trypsin-based detachment assay measuring time required to detach cells from substrate48. PARP14 knockdown cells were more resistant to trypsinization than controls, with ~ 2 times more cells remaining adhered after similar periods of trypsinization (Figure 6A). Second we employed a centrifugation assay comparing the number of cells remaining adhered to substrate after constant application of centrifugal force. Approximately 5 times more PARP14 knock-down cells remained adhered to substrate after centrifugation compared to controls (Figure 6B). Third, we used a cell-spreading assay measuring the area of cell spreading per unit time (Figure 6C). At early time points when cells adhered to substrate but had not yet formed defined FAs, control and PARP14 knock-downs were of similar size suggesting that the morphological defects observed in PARP14 knock-downs were dependent on FA formation and not simply a property of the cells themselves. As cells assembled FAs and spread onto substrate, PARP14 knock-down cells covered ~twice the surface area of control cells at each time point (Figure 6C). These three independent assays suggest that the adhesive strength between FAs and substrate is stronger in PARP14 knock-downs relative to control. It is likely that this increased adhesive strength leads to slowed FA disassembly and prevents cellular protrusions from properly retracting.

Bottom Line: These include the regulation of membrane structures, cell viability, cell division and the actin cytoskeleton.Further analysis of PARP14 shows that it is a component of focal adhesion complexes required for proper cell motility and focal adhesion function.In total, we show that PARP proteins are critical regulators of eukaryotic physiology.

View Article: PubMed Central - PubMed

Affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

ABSTRACT
The poly(ADP-ribose) polymerase (PARP) family of proteins use NAD(+) as their substrate to modify acceptor proteins with ADP-ribose modifications. The function of most PARPs under physiological conditions is unknown. Here, to better understand this protein family, we systematically analyse the cell cycle localization of each PARP and of poly(ADP-ribose), a product of PARP activity, then identify the knockdown phenotype of each protein and perform secondary assays to elucidate function. We show that most PARPs are cytoplasmic, identify cell cycle differences in the ratio of nuclear to cytoplasmic poly(ADP-ribose) and identify four phenotypic classes of PARP function. These include the regulation of membrane structures, cell viability, cell division and the actin cytoskeleton. Further analysis of PARP14 shows that it is a component of focal adhesion complexes required for proper cell motility and focal adhesion function. In total, we show that PARP proteins are critical regulators of eukaryotic physiology.

Show MeSH
Related in: MedlinePlus