The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

Abstract

Organelle distribution is regulated over the course of the cell cycle to ensure that each of the cells produced at the completion of division inherits a full complement of organelles. In yeast, the protein Num1 functions in the positioning and inheritance of two essential organelles, mitochondria and the nucleus. Specifically, Num1 anchors mitochondria as well as dynein to the cell cortex, and this anchoring activity is required for proper mitochondrial distribution and dynein-mediated nuclear inheritance. The assembly of Num1 into clusters at the plasma membrane is critical for both of its anchoring functions. We have previously shown that mitochondria drive the assembly of Num1 clusters and that these mitochondria-assembled Num1 clusters serve as cortical attachment sites for dynein. Here we further examine the role for mitochondria in dynein anchoring. Using a GFP-αGFP nanobody targeting system, we synthetically clustered Num1 on eisosomes to bypass the requirement for mitochondria in Num1 cluster formation. Utilizing this system, we found that mitochondria positively impact the ability of synthetically clustered Num1 to anchor dynein and support dynein function even when mitochondria are no longer required for cluster formation. Thus, the role of mitochondria in regulating dynein function extends beyond simply concentrating Num1; mitochondria likely promote an arrangement of Num1 within a cluster that is competent for dynein anchoring. This functional dependency between mitochondrial and nuclear positioning pathways likely serves as a mechanism to order and integrate major cellular organization systems over the course of the cell cycle.

Keywords: Mitochondrial positioning; dynein; inter-organelle contact sites.

Figure 1.

A system to synthetically cluster Num1 at the cell cortex. (A and B) The GFP-αGFP nanobody targeting system used to synthetically cluster Num1 on the plasma membrane. The schematic on the left in A depicts a wildtype Num1 cluster, which assembles only in the presence of mitochondria. The schematic on the right in A depicts mitochondria-independent assembly of a synthetic Num1 cluster driven by the GFP-αGFP nanobody targeting system. Schematics of constructs used in the study are shown in B. CC, coiled-coil domain; EF, EF hand-like domain; mito, mitochondria; PAN, Pil1-associated Num1; PH, pleckstrin homology domain; PM, plasma membrane; αGFP, αGFP nanobody. (C) Cells expressing Pil1, Num1, Num1ΔPH, PAN, Num1ΔCCΔPH, or PANΔCC as GFP fusions were analyzed by fluorescence microscopy. Deconvolved, maximum intensity, whole cell Z-projections are shown. The cell cortex is outlined with a dashed white line. Bar, 2 µm. Quantification of the total number of clusters per cell is shown as the mean ± SD indicated. n = the number of cells quantified, as indicated in the figure. (D) Cells expressing Lsp1-mKate and Pil1, Num1, or PAN as GFP fusions were analyzed by fluorescence microscopy. Single focal planes of deconvolved Z-stacks are shown. The cell cortex is outlined with a dashed white line. Bar, 2 µm. The Pearson’s correlation between Lsp1 and the indicated GFP fusion is shown as the mean ± SD of 10 fields of view with at least 15 cells per field.

Figure 2.

The relationship between PAN clusters and mitochondria. (A) Cells expressing Num1, Num1ΔPH, PAN, Num1ΔCCΔPH, or PANΔCC as GFP fusions along with mitoRED were analyzed by fluorescence microscopy. Deconvolved, maximum intensity, whole cell Z-projections are shown. The cell cortex is outlined with a dashed white line. Bar, 2 µm. (B) Quantification of the number of mitochondria-associated clusters per cell in cells expressing Num1, PAN, and PANΔCC along with mitoRED. To be considered a mitochondria-associated cluster, mitochondria had to remain associated with a cluster for ≥ 1.5 min. Each dot represents a cell. The mean ± SD are shown. n = the number of cells quantified, as indicated in the figure. (C) A plot of the number of mitochondria-associated Num1 and PAN clusters per cell versus the total number of clusters per cell. The color of each rectangle represents the number of cells, as indicated by the key, with that number of total and mitochondria-associated clusters. n = the number of cells quantified, as indicated in the figure. (D) Mitochondria-associated Num1, PAN, and PANΔCC clusters per cell as a percentage of total clusters per cell. n = 3 independent experiments in which ≥ 50 cells were counted. The mean ± SD is shown. (E) To assess growth, serial dilutions of the indicated strains were plated on rich medium and grown at the permissive (24°C) or non-permissive (37°C) temperature for the temperature sensitive fzo1-1 allele, as indicated.

Figure 3.

PAN clusters support dynein function. (A) To assess growth, serial dilutions of the indicated strains were plated on rich medium and grown at 30°C. (B-C) Wildtype W303, Δdyn1, Δnum1, num1ΔPH, and PAN cells expressing Ruby-Tub1 were visualized by fluorescence microscopy. Representative, deconvolved, maximum intensity, whole cell Z-projections of cells with spindles scored as correctly oriented and misoriented are shown (B). The cell cortex is outlined with a dashed line. Bars, 2 μm. Quantification of the percent of cells with misoriented spindles is shown as the mean ± SD (C). Large buds with spindles > 1.25 μm were scored. n = 3 independent experiments of ≥ 52 spindles for each experiment. The dashed line represents the mean wildtype spindle misorientation value. p values are in comparison to wildtype W303 unless otherwise denoted by brackets. *** p < 0.001, ** p < 0.01, * p < 0.02; n.s., not significant. (D) Misoriented spindles in Δkar9, Δkar9 num1ΔPH, and Δkar9 PAN were quantified as described in C. The dashed line represents the mean wildtype spindle misorientation value from panel C. p values are in comparison to Δkar9 unless otherwise denoted by brackets. *** p < 0.001; n.s., not significant.

Figure 4.

Mitochondria-associated PAN clusters anchor dynein. (A) NUM1-yEGFP or PAN cells expressing Dyn1-mKate and mitoBFP were visualized by fluorescence microscopy over time. Single focal planes of deconvolved Z-stacks are shown. Arrows indicate a colocalization event. The cell cortex is outlined with a dashed line. Bars, 2 μm. Time is in minutes. (B) Quantification of the percent of cortical dynein foci that colocalize with mitochondria. n ≥ 103 cortical dynein foci. Standard error of proportion is shown. To be considered cortically anchored, a Dyn1-mKate focus had to remain stable at the cell cortex for ≥ 1.5 minutes. (C) mito^AID NUM1-yEGFP and mito^AID PAN cells expressing mitoRED were grown in the absence or presence of auxin and visualized by fluorescence microscopy. Deconvolved, maximum intensity, whole cell Z-projections are shown. The cell cortex is outlined with a dashed white line. Bar, 2 µm. (D) mito^AID NUM1-yEGFP, mito^AID num1ΔPH, and mito^AID PAN cells expressing Ruby-Tub1 and mitoBFP were visualized by fluorescence microscopy. Misoriented spindles were quantified as described in Figure 3(c). n = 3 independent experiments of ≥ 46 spindles for each experiment. The mean ± SD is shown. p values are in comparison to wildtype NUM1 cells, and p values in parentheses are in comparison to the identical genotype in the absence of auxin. *** p < 0.001, ** p < 0.01; n.s., not significant. (E) Model: The anchoring of dynein at the cell cortex by Num1 clusters assembled in the absence of mitochondria using the GFP-αGFP nanobody targeting system is positively influenced by the presence of mitochondria at a cluster.