Abstract:An N-terminal BAR-like domain in Num1 mediates its assembly into patches and its interaction with dynein to promote spindle translocation into the mitotic yeast bud.
“…S1A). These data, which are consistent with previous studies (17), indicate that the Num1-mitochondria interaction is independent of mitochondrial division and fusion and of Num1's role in nuclear migration, respectively. Together the localization of Num1 clusters, their association with mitochondria, and the behavior of mitochondria in their proximity suggest that Num1 functions to tether mitochondria to the cortex physically to ensure accurate distribution of the organelle between mother and daughter cells.…”
Section: Num1 Is Essential In δFzo1 δDnm1supporting
To elucidate the functional roles of mitochondrial dynamics in vivo, we identified genes that become essential in cells lacking the dynamin-related proteins Fzo1 and Dnm1, which are required for mitochondrial fusion and division, respectively. The screen identified Num1, a cortical protein implicated in mitochondrial division and distribution that also functions in nuclear migration. Our data indicate that Num1, together with Mdm36, forms a physical tether that robustly anchors mitochondria to the cell cortex but plays no direct role in mitochondrial division. Our analysis indicates that Num1-dependent anchoring is essential for distribution of the static mitochondrial network in fzo1 dnm1 cells. Consistently, expression of a synthetic mitochondria-cortex tether rescues the viability of fzo1 dnm1 num1 cells. We find that the cortical endoplasmic reticulum (ER) also is a constituent of the Num1 mitochondria-cortex tether, suggesting an active role for the ER in mitochondrial positioning in cells. Thus, taken together, our findings identify Num1 as a key component of a mitochondria-ER-cortex anchor, which we termed "MECA," that functions in parallel with mitochondrial dynamics to distribute and position the essential mitochondrial network.T he shape and cellular distribution of mitochondria depend on the integrated and regulated activities of mitochondrial division and fusion, motility, and tethering (1). A key question is how these mitochondrial behaviors are coordinated to shape and position mitochondria properly in response to the changing needs of the cell. To begin to address this question, an understanding of the molecular basis of mitochondrial behaviors is essential.The molecular mechanisms underlying mitochondrial division and fusion are best understood in terms of mitochondrial behaviors (1, 2). At the heart of the molecular machines that mediate mitochondrial division and fusion are dynamin-related proteins (DRPs) that function via GTP-dependent self-assembly and GTP hydrolysismediated conformational changes to remodel membranes. The DRP Dnm1/DRP1 (in yeast and mammals, respectively) drives the scission of mitochondrial membranes, and the DRPs Fzo1/ MFN1/2 and Mgm1/OPA1 mediate fusion of the outer and inner mitochondrial membranes, respectively. The relative rates of mitochondrial division and fusion are major determinants of the steadystate structure of the organelle and greatly influence its distribution. Attenuation of mitochondrial division leads to a more interconnected, collapsed, and less distributable mitochondrial network, and attenuation of mitochondrial fusion results in mitochondrial fragmentation and pronounced defects in the transmission and distribution of mtDNA.Although mitochondrial division and fusion are important, additional parallel pathways also are likely to be important for mitochondrial distribution. For example, the stable positioning of mitochondria at specific cellular locations, an indication of active tethering mechanisms, has been observed in many different cell types. In ...
“…S1A). These data, which are consistent with previous studies (17), indicate that the Num1-mitochondria interaction is independent of mitochondrial division and fusion and of Num1's role in nuclear migration, respectively. Together the localization of Num1 clusters, their association with mitochondria, and the behavior of mitochondria in their proximity suggest that Num1 functions to tether mitochondria to the cortex physically to ensure accurate distribution of the organelle between mother and daughter cells.…”
Section: Num1 Is Essential In δFzo1 δDnm1supporting
To elucidate the functional roles of mitochondrial dynamics in vivo, we identified genes that become essential in cells lacking the dynamin-related proteins Fzo1 and Dnm1, which are required for mitochondrial fusion and division, respectively. The screen identified Num1, a cortical protein implicated in mitochondrial division and distribution that also functions in nuclear migration. Our data indicate that Num1, together with Mdm36, forms a physical tether that robustly anchors mitochondria to the cell cortex but plays no direct role in mitochondrial division. Our analysis indicates that Num1-dependent anchoring is essential for distribution of the static mitochondrial network in fzo1 dnm1 cells. Consistently, expression of a synthetic mitochondria-cortex tether rescues the viability of fzo1 dnm1 num1 cells. We find that the cortical endoplasmic reticulum (ER) also is a constituent of the Num1 mitochondria-cortex tether, suggesting an active role for the ER in mitochondrial positioning in cells. Thus, taken together, our findings identify Num1 as a key component of a mitochondria-ER-cortex anchor, which we termed "MECA," that functions in parallel with mitochondrial dynamics to distribute and position the essential mitochondrial network.T he shape and cellular distribution of mitochondria depend on the integrated and regulated activities of mitochondrial division and fusion, motility, and tethering (1). A key question is how these mitochondrial behaviors are coordinated to shape and position mitochondria properly in response to the changing needs of the cell. To begin to address this question, an understanding of the molecular basis of mitochondrial behaviors is essential.The molecular mechanisms underlying mitochondrial division and fusion are best understood in terms of mitochondrial behaviors (1, 2). At the heart of the molecular machines that mediate mitochondrial division and fusion are dynamin-related proteins (DRPs) that function via GTP-dependent self-assembly and GTP hydrolysismediated conformational changes to remodel membranes. The DRP Dnm1/DRP1 (in yeast and mammals, respectively) drives the scission of mitochondrial membranes, and the DRPs Fzo1/ MFN1/2 and Mgm1/OPA1 mediate fusion of the outer and inner mitochondrial membranes, respectively. The relative rates of mitochondrial division and fusion are major determinants of the steadystate structure of the organelle and greatly influence its distribution. Attenuation of mitochondrial division leads to a more interconnected, collapsed, and less distributable mitochondrial network, and attenuation of mitochondrial fusion results in mitochondrial fragmentation and pronounced defects in the transmission and distribution of mtDNA.Although mitochondrial division and fusion are important, additional parallel pathways also are likely to be important for mitochondrial distribution. For example, the stable positioning of mitochondria at specific cellular locations, an indication of active tethering mechanisms, has been observed in many different cell types. In ...
“…PAN clusters were more abundant than clusters formed by wildtype Num1 (Figure 1(c)), which is consistent with the idea that targeting Num1ΔPH to eisosomes drives cluster formation in a manner distinct from wildtype Num1. Consistently, in contrast to wildtype Num1 cluster formation which depends on the CC domain of the protein [10,11], PAN clusters were no longer dependent on the CC domain as PANΔCC formed clusters indistinguishable from PAN (Figure 1(c)). Together, these results indicate that Pil1-αGFP is able to target and synthetically cluster Num1ΔPH on the plasma membrane.10.1080/15384101.2018.1480226-F0001Figure 1. A system to synthetically cluster Num1 at the cell cortex .…”
Section: Resultssupporting
confidence: 53%
“…The PH domain, which binds with high specificity to PI 4,5 P 2 , targets Num1 to the plasma membrane [13,14], where the protein is found in clusters as well as in a pool that is diffusely localized along the plasma membrane and with cortical ER [3,5,10,15,16]. Cluster formation, which is dependent on the CC domain and an interaction with mitochondria (Figure 1(a)), is critical for the function of Num1 in both mitochondria and dynein anchoring [10,11,17]. Cluster formation has been proposed to enhance the interaction between Num1 and its membrane and protein binding partners and, consequently, the ability of Num1 to robustly tether mitochondria and dynein to the cell cortex [10,11].…”
Section: Introductionmentioning
confidence: 99%
“…Cluster formation, which is dependent on the CC domain and an interaction with mitochondria (Figure 1(a)), is critical for the function of Num1 in both mitochondria and dynein anchoring [10,11,17]. Cluster formation has been proposed to enhance the interaction between Num1 and its membrane and protein binding partners and, consequently, the ability of Num1 to robustly tether mitochondria and dynein to the cell cortex [10,11]. In addition, Num1 is proposed to directly activate dynein [18,19].…”
Section: Introductionmentioning
confidence: 99%
“…While dynein is not required for Num1-mediated mitochondrial anchoring [10,11], mitochondria drive the assembly of Num1 clusters that serve as cortical attachment sites for dynein [17]. These results raise the question of whether the role for mitochondria in dynein anchoring is to simply concentrate Num1 or whether mitochondria play an additional role in driving the assembly of a structure competent to tether dynein.…”
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.
Cytoplasmic dynein is the major minus-end-directed motor protein in eukaryotes, and has functions ranging from organelle and vesicle transport to spindle positioning and orientation. The mode of regulation of dynein in the cell remains elusive, but a tantalising possibility is that dynein is maintained in an inhibited, non-motile state until bound to cargo. In vivo, stable attachment of dynein to the cell membrane via anchor proteins enables dynein to produce force by pulling on microtubules and serves to organise the nuclear material. Anchor proteins of dynein assume diverse structures and functions and differ in their interaction with the membrane. In yeast, the anchor protein has come to the fore as one of the key mediators of dynein activity. In other systems, much is yet to be discovered about the anchors, but future work in this area will prove invaluable in understanding dynein regulation in the cell.
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