Membrane contact sites (MCSs) facilitate communication and organization of organelles that contribute to fundamental cellular processes. It has become increasingly appreciated how extensive and essential MCSs are for cellular health, and this field is rapidly growing. We use budding yeast as a model system to study the mechanisms of how polarized cells establish, organize, and regulate contacts between organelles. Using the facile yeast system will help us better understand these complex mechanisms in a simpler system to provide the framework for understanding MCS regulation in high eukaryotes.The mitochondria-ER cortex anchor (MECA) is a budding yeast MCS that facilitates the interaction of mitochondria with the plasma membrane (PM) at sites of the cortical endoplasmic reticulum (ER). MECA also serves as a cortical attachment site for the molecular motor protein dynein, which provides the force for nuclear positioning and migration. The main protein component of MECA Num1, contains two lipid interacting domains to bridge the interaction between mitochondria and the PM. It was previously shown that mitochondria drive the assembly of cortical Num1 clusters, and that dynein can be simultaneously anchored at mitochondria tethering sites. This thesis investigates the interplay and regulation of MECA’s roles in mitochondrial tethering and dynein anchoring. We developed an innovative synthetic clustering system to separate mitochondria tethering from cluster formation to study dynein regulation. We find that dynein preferentially interacts with mitochondria-associated synthetic clusters. Furthermore, when we conditionally inhibit mitochondrial inheritance, we observe defects in dynein function. These data suggest that mitochondria bias the anchoring of dynein and that mitochondria may impact Num1 cluster arrangement. To further understand the regulation of dynein anchoring at MECA, I used a structure function approach in combination with our synthetic clustering system to identify the domain of Num1 that was important for mitochondria-associated dynein anchoring. I find that the EF hand-like motif (EFLM) of Num1 is important for the integration of mitochondria tethering and dynein anchoring. When the EFLM is disrupted in our synthetic system, mitochondria no longer bias the anchoring of dynein. Additionally, in full length Num1 with the EFLM disrupted, dynein function is no longer dependent on mitochondrial inheritance. These data suggest that dynein processivity is not dependent on mitochondria, but the EFLM is important to couple mitochondria tethering with dynein anchoring. We believe this is a way for the cell to ensure that mitochondrial inheritance occurs prior to nuclear inheritance, acting as a surveillance system that contributes to the order of organelle inheritance. Overall, these findings contribute to our understanding of the regulation and functions of a mitochondrial membrane contact site.

Creator

• Anderson, Heidi Lynn

DOI

• https://doi.org/10.21985/n2-8d2d-wh25

Subject

• Cellular biology

Language

• en

Alternate Identifier

• etdadmin_upload_873821
• http://dissertations.umi.com/northwestern:15900

Keyword

• Membrane Contact Sites
• Organelle Positioning
• Mitochondria

Date created

• 2022-01-01

Resource type

• Dissertation

Rights statement

• In Copyright