Limits...
A tether for Woronin body inheritance is associated with evolutionary variation in organelle positioning.

Ng SK, Liu F, Lai J, Low W, Jedd G - PLoS Genet. (2009)

Bottom Line: In most species, WBs are tethered directly to the pore rim, however, Neurospora and relatives have evolved a delocalized pattern of cortex association.Using a new method for the construction of chromosomally encoded fusion proteins, marker fusion tagging (MFT), we show that a LAH-1/LAH-2 fusion can reproduce the ancestral pattern in Neurospora.Our results identify the link between the WB and cell cortex and suggest that splitting of leashin played a key role in the adaptive evolution of organelle localization.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore.

ABSTRACT
Eukaryotic organelles evolve to support the lifestyle of evolutionarily related organisms. In the fungi, filamentous Ascomycetes possess dense-core organelles called Woronin bodies (WBs). These organelles originate from peroxisomes and perform an adaptive function to seal septal pores in response to cellular wounding. Here, we identify Leashin, an organellar tether required for WB inheritance, and associate it with evolutionary variation in the subcellular pattern of WB distribution. In Neurospora, the leashin (lah) locus encodes two related adjacent genes. N-terminal sequences of LAH-1 bind WBs via the WB-specific membrane protein WSC, and C-terminal sequences are required for WB inheritance by cell cortex association. LAH-2 is localized to the hyphal apex and septal pore rim and plays a role in colonial growth. In most species, WBs are tethered directly to the pore rim, however, Neurospora and relatives have evolved a delocalized pattern of cortex association. Using a new method for the construction of chromosomally encoded fusion proteins, marker fusion tagging (MFT), we show that a LAH-1/LAH-2 fusion can reproduce the ancestral pattern in Neurospora. Our results identify the link between the WB and cell cortex and suggest that splitting of leashin played a key role in the adaptive evolution of organelle localization.

Show MeSH

Related in: MedlinePlus

N-terminal sequences of LAH associate with WBs via C-terminal sequences of WSC.(A) The first 344 amino acids of LAH co-localize with WSC at the WB surface. LAH1–344-RFP was co-expressed with WSC-GFP and visualized by confocal microscopy. Bar = 2 µm. (B) LAH1–344 association with a dense organellar fraction depends on the presence of WSC. LAH1–344-HA was expressed in wild-type and wsc mutant background and a crude organellar fraction (T) was separated into supernatant (S) and pellet (P) fractions and the distribution of LAH1–344-HA was revealed with anti-HA epitope antibodies. (C) C-terminal tail of WSC is required for WB-segregation but not the production of nascent WBs. HEX assembly distribution was assessed in a strain expressing WSCΔC-GFP. Inset shows the distribution of WSCΔC-GFP and RFP-PTS1, which reveals the peroxisome matrix. (D) LAH1–344 does not associate with the WB in the WSCΔC-GFP expressing strain. WSCΔC-GFP and LAH1–344-RFP were co-expressed and visualized as in (A). Bar = 2 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2690989&req=5

pgen-1000521-g003: N-terminal sequences of LAH associate with WBs via C-terminal sequences of WSC.(A) The first 344 amino acids of LAH co-localize with WSC at the WB surface. LAH1–344-RFP was co-expressed with WSC-GFP and visualized by confocal microscopy. Bar = 2 µm. (B) LAH1–344 association with a dense organellar fraction depends on the presence of WSC. LAH1–344-HA was expressed in wild-type and wsc mutant background and a crude organellar fraction (T) was separated into supernatant (S) and pellet (P) fractions and the distribution of LAH1–344-HA was revealed with anti-HA epitope antibodies. (C) C-terminal tail of WSC is required for WB-segregation but not the production of nascent WBs. HEX assembly distribution was assessed in a strain expressing WSCΔC-GFP. Inset shows the distribution of WSCΔC-GFP and RFP-PTS1, which reveals the peroxisome matrix. (D) LAH1–344 does not associate with the WB in the WSCΔC-GFP expressing strain. WSCΔC-GFP and LAH1–344-RFP were co-expressed and visualized as in (A). Bar = 2 µm.

Mentions: LAH should localize to the WB surface if it plays a direct role in WB segregation. We next assessed localization of LAH fragments of fused to the red fluorescent protein (RFP) and identified an N-terminal domain encompassing amino acids 1–344 (LAH1–344RFP) that co-localizes with WSC-GFP at the WB surface (Figure 3A). In extracts prepared from cells expressing an HA-epitope tagged version of this LAH fragment, the fusion protein sediments at very low centrifugal forces, consistent with association with the dense-core WB, but is rendered mostly soluble in extracts prepared from a wsc deletion strain (Figure 3B), suggesting that LAH associates with WBs via WSC. To further investigate this interaction, we examined WSC deletion mutants; deletion of the WSC C-terminal tail (Δ236–307) blocks WB segregation, but not the envelopment of HEX assemblies and production of nascent WBs (Figure 3C). WB localization of LAH1–344RFP is also abolished in the WSC C-terminal deletion, further suggesting that N-terminal sequences of LAH associate with WBs through the C-terminus of WSC (Figure 3D).


A tether for Woronin body inheritance is associated with evolutionary variation in organelle positioning.

Ng SK, Liu F, Lai J, Low W, Jedd G - PLoS Genet. (2009)

N-terminal sequences of LAH associate with WBs via C-terminal sequences of WSC.(A) The first 344 amino acids of LAH co-localize with WSC at the WB surface. LAH1–344-RFP was co-expressed with WSC-GFP and visualized by confocal microscopy. Bar = 2 µm. (B) LAH1–344 association with a dense organellar fraction depends on the presence of WSC. LAH1–344-HA was expressed in wild-type and wsc mutant background and a crude organellar fraction (T) was separated into supernatant (S) and pellet (P) fractions and the distribution of LAH1–344-HA was revealed with anti-HA epitope antibodies. (C) C-terminal tail of WSC is required for WB-segregation but not the production of nascent WBs. HEX assembly distribution was assessed in a strain expressing WSCΔC-GFP. Inset shows the distribution of WSCΔC-GFP and RFP-PTS1, which reveals the peroxisome matrix. (D) LAH1–344 does not associate with the WB in the WSCΔC-GFP expressing strain. WSCΔC-GFP and LAH1–344-RFP were co-expressed and visualized as in (A). Bar = 2 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2690989&req=5

pgen-1000521-g003: N-terminal sequences of LAH associate with WBs via C-terminal sequences of WSC.(A) The first 344 amino acids of LAH co-localize with WSC at the WB surface. LAH1–344-RFP was co-expressed with WSC-GFP and visualized by confocal microscopy. Bar = 2 µm. (B) LAH1–344 association with a dense organellar fraction depends on the presence of WSC. LAH1–344-HA was expressed in wild-type and wsc mutant background and a crude organellar fraction (T) was separated into supernatant (S) and pellet (P) fractions and the distribution of LAH1–344-HA was revealed with anti-HA epitope antibodies. (C) C-terminal tail of WSC is required for WB-segregation but not the production of nascent WBs. HEX assembly distribution was assessed in a strain expressing WSCΔC-GFP. Inset shows the distribution of WSCΔC-GFP and RFP-PTS1, which reveals the peroxisome matrix. (D) LAH1–344 does not associate with the WB in the WSCΔC-GFP expressing strain. WSCΔC-GFP and LAH1–344-RFP were co-expressed and visualized as in (A). Bar = 2 µm.
Mentions: LAH should localize to the WB surface if it plays a direct role in WB segregation. We next assessed localization of LAH fragments of fused to the red fluorescent protein (RFP) and identified an N-terminal domain encompassing amino acids 1–344 (LAH1–344RFP) that co-localizes with WSC-GFP at the WB surface (Figure 3A). In extracts prepared from cells expressing an HA-epitope tagged version of this LAH fragment, the fusion protein sediments at very low centrifugal forces, consistent with association with the dense-core WB, but is rendered mostly soluble in extracts prepared from a wsc deletion strain (Figure 3B), suggesting that LAH associates with WBs via WSC. To further investigate this interaction, we examined WSC deletion mutants; deletion of the WSC C-terminal tail (Δ236–307) blocks WB segregation, but not the envelopment of HEX assemblies and production of nascent WBs (Figure 3C). WB localization of LAH1–344RFP is also abolished in the WSC C-terminal deletion, further suggesting that N-terminal sequences of LAH associate with WBs through the C-terminus of WSC (Figure 3D).

Bottom Line: In most species, WBs are tethered directly to the pore rim, however, Neurospora and relatives have evolved a delocalized pattern of cortex association.Using a new method for the construction of chromosomally encoded fusion proteins, marker fusion tagging (MFT), we show that a LAH-1/LAH-2 fusion can reproduce the ancestral pattern in Neurospora.Our results identify the link between the WB and cell cortex and suggest that splitting of leashin played a key role in the adaptive evolution of organelle localization.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore.

ABSTRACT
Eukaryotic organelles evolve to support the lifestyle of evolutionarily related organisms. In the fungi, filamentous Ascomycetes possess dense-core organelles called Woronin bodies (WBs). These organelles originate from peroxisomes and perform an adaptive function to seal septal pores in response to cellular wounding. Here, we identify Leashin, an organellar tether required for WB inheritance, and associate it with evolutionary variation in the subcellular pattern of WB distribution. In Neurospora, the leashin (lah) locus encodes two related adjacent genes. N-terminal sequences of LAH-1 bind WBs via the WB-specific membrane protein WSC, and C-terminal sequences are required for WB inheritance by cell cortex association. LAH-2 is localized to the hyphal apex and septal pore rim and plays a role in colonial growth. In most species, WBs are tethered directly to the pore rim, however, Neurospora and relatives have evolved a delocalized pattern of cortex association. Using a new method for the construction of chromosomally encoded fusion proteins, marker fusion tagging (MFT), we show that a LAH-1/LAH-2 fusion can reproduce the ancestral pattern in Neurospora. Our results identify the link between the WB and cell cortex and suggest that splitting of leashin played a key role in the adaptive evolution of organelle localization.

Show MeSH
Related in: MedlinePlus