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. 2014 Jan 1;127(Pt 1):250-7.
doi: 10.1242/jcs.140996. Epub 2013 Nov 4.

Golgi enlargement in Arf-depleted yeast cells is due to altered dynamics of cisternal maturation

Madhura Bhave  1 , Effrosyni Papanikou, Prasanna Iyer, Koushal Pandya, Bhawik Kumar Jain, Abira Ganguly, Chandrakala Sharma, Ketakee Pawar, Jotham Austin 2nd, Kasey J Day, Olivia W Rossanese, Benjamin S Glick, Dibyendu Bhattacharyya
Affiliations

Golgi enlargement in Arf-depleted yeast cells is due to altered dynamics of cisternal maturation

Madhura Bhave et al. J Cell Sci. .

Abstract

Regulation of the size and abundance of membrane compartments is a fundamental cellular activity. In Saccharomyces cerevisiae, disruption of the ADP-ribosylation factor 1 (ARF1) gene yields larger and fewer Golgi cisternae by partially depleting the Arf GTPase. We observed a similar phenotype with a thermosensitive mutation in Nmt1, which myristoylates and activates Arf. Therefore, partial depletion of Arf is a convenient tool for dissecting mechanisms that regulate Golgi structure. We found that in arf1Δ cells, late Golgi structure is particularly abnormal, with the number of late Golgi cisternae being severely reduced. This effect can be explained by selective changes in cisternal maturation kinetics. The arf1Δ mutation causes early Golgi cisternae to mature more slowly and less frequently, but does not alter the maturation of late Golgi cisternae. These changes quantitatively explain why late Golgi cisternae are fewer in number and correspondingly larger. With a stacked Golgi, similar changes in maturation kinetics could be used by the cell to modulate the number of cisternae per stack. Thus, the rates of processes that transform a maturing compartment can determine compartmental size and copy number.

Keywords: Arf; Cisternal maturation; Golgi; Organelle number; Organelle size.

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Figures

Fig. 1.
Fig. 1.
A conditional mutation in NMT1 generates enlarged late Golgi cisternae. (A) Representative images of the wild-type (WT) parental strain, the thermosensitive mutant, and the mutant transformed with a centromeric plasmid encoding NMT1. The plasma membrane was labeled with mCherry–Ras2 (red), and the late Golgi was labeled with Sec7–GFPx3 (green). The top row shows projected confocal sections of a central portion of the cell, and the bottom row shows 3D renderings. Scale bar: 1 µm. (B,C) Images of the type shown in A were quantified by rendering late Golgi cisternae as closed surfaces. The following strains were examined: the wild-type, the thermosensitive mutant, the mutant in which the nmt1 T400I mutation was reverted by gene replacement, the mutant transformed with a centromeric plasmid encoding NMT1, the mutant transformed with the plasmid encoding NMT1 and then cured of this plasmid (indicated by the strikethrough), the mutant transformed with a high copy number plasmid encoding ARF1, and the mutant transformed with the plasmid encoding ARF1 and then cured of this plasmid (indicated by the strikethrough). Sizes of the rendered late Golgi elements were quantified by measuring either (B) the average volume or (C) the average X–Y-plane diameter. Error bars indicate s.e.m.
Fig. 2.
Fig. 2.
The arf1Δ mutation generates enlarged late Golgi cisternae that label with FM 4-64. (A) arf1Δ cells were imaged by fluorescence microscopy as in Fig. 1 to visualize the plasma membrane (red) and late Golgi (green). Scale bar: 1 µm. (B) The average volume and average X–Y-plane diameter of late Golgi cisternae were measured as in Fig. 1 for wild-type and arf1Δ strains. Error bars indicate s.e.m. (C) Wild-type or arf1Δ cells expressing Sec7-6xGFP were grown in SD medium at 23°C to an OD600 of 0.6, then incubated with 0.8 µM FM 4-64 for 5 minutes. SCAS was added to 4 µM, and cells were compressed beneath a coverslip. Confocal images were captured ∼2.5 minutes after SCAS addition. Scale bar: 1 µm. (D) Wild-type or arf1Δ cells expressing Sec7-GFPx3 were rapidly frozen, then freeze substituted and embedded in plastic. Left: thick sections were imaged by fluorescence microscopy to identify late Golgi cisternae. Scale bars: 2 µm. Middle: the same sections were analyzed by electron tomography to visualize membranes in tomographic slices. Scale bars: 0.5 µm. Right: membrane contours were traced to model Golgi cisternae (green, blue, yellow) and the plasma membrane (purple). Scale bars: 0.5 µm.
Fig. 3.
Fig. 3.
The arf1Δ mutation differentially affects the early and late Golgi. (A) The average numbers of early Golgi cisternae, marked with GFP–Vrg4, or late Golgi cisternae, marked with Sec7–DsRed, in wild-type and arf1Δ strains. Error bars indicate s.e.m. (B) A 10-minute 4D movie was generated for an arf1Δ cell expressing Sec7-3xGFP. Fluorescence intensities were quantified for each cisterna that appeared and subsequently disappeared during the recording. The different colors represent distinct cisternae. (C) The average frequency of early-to-late Golgi transitions as indicated by loss of GFP–Vrg4 and acquisition of Sec7–DsRed in wild-type and arf1Δ strains. Error bars indicate s.e.m. (D) The average persistence time on cisternae of the early Golgi marker GFP–Vrg4 and the late Golgi marker Sec7–DsRed in wild-type and arf1Δ strains. Error bars indicate s.e.m.
Fig. 4.
Fig. 4.
Golgi cisternae can undergo homotypic fusion. (A) Frames from a 4D movie (supplementary material Movie 4) illustrate a homotypic fusion event in an arf1Δ cell. The arrowheads mark two GFP–Vrg4-labeled early Golgi cisternae that fused before maturing into a Sec7–DsRed-labeled late Golgi cisterna. Time is shown in minutes:seconds. Scale bar: 1 µm. (B) The average number of homotypic fusion events per minute for early and late Golgi cisternae in wild-type and arf1Δ strains.
Fig. 5.
Fig. 5.
Changes in maturation kinetics could alter the number of cisternae in a stacked Golgi. A Golgi stack is represented as consisting of two classes of cisternae: the Golgi itself (green) and the trans-Golgi network (TGN; orange). These classes would correspond in S. cerevisiae to the early and late Golgi, respectively. If the persistence time of Golgi cisternae were increased without changing other parameters, the stack would contain more Golgi cisternae but the same number of TGN cisternae.
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