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Research

The following is an overview of the types of questions the lab is interested in studying:

 

1.) How does inner nuclear composition change during developmental transitions? Are inner nuclear membrane proteins removed or degraded?

 

2.) How is the nuclear envelope remodeled during mitosis and meiosis in different organisms? How does the nuclear membrane interact with itself and other organelles?

 

3.) What sets the size of the nucleus and the spindle? Are these coupled during closed mitosis?

 

4.) How are nuclear components partitioned during cell division? How are nuclear pore complexes distributed on the nuclear membrane?

 

How does inner nuclear composition change during developmental transitions? Are inner nuclear membrane proteins removed or degraded?

Changes in the composition of the inner nuclear membrane occur during development and differentiation. However, how the inner nuclear membrane is altered and how changes in its proteome are coupled with nuclear function are poorly understood, in part because it is difficult to assay inner nuclear membrane composition in cells.

 

To overcome this obstacle, we developed a system using split-GFP that enabled us to systematically study inner nuclear membrane localization of endogenously expressed proteins in live cells.  We used this system to determine the inner nuclear membrane proteome of mitotic yeast cells. 

 

We can now leverage the power of yeast genetics to address important questions about inner nuclear membrane regulation and homeostasis. Over half of our inner nuclear membrane hits have a direct human ortholog so this work will help understand how inner nuclear membrane protein defects contribute to cancer, aging and other diseases.


MYTH

 

The use of split-GFP to study nuclear envelope compartments. Adapted from Smoyer et al. JCB 2016.



How is the nuclear envelope remodeled during mitosis and meiosis in different organisms? How does the nuclear membrane interact with itself and other organelles?

How do cytoplasmic factors required for chromosome segregation access DNA inside the nucleus? Eukaryotic cells have an array of answers to this basic question of cell division, each with distinct advantages and disadvantages. Most higher eukaryotes fragment the nuclear envelope upon mitotic entry, leading to rapid spindle formation. However, this strategy exposes cells to potential defects during re-compartmentalization, which could result in the formation of micronuclei and aneuploidy.

 

Rapidly dividing cells, such as single celled eukaryotes or embryos, dismantle only small regions of the nuclear envelope. This allows microtubule-organizing centers, known as centrosomes in metazoans and spindle pole bodies in yeast, to form a mitotic spindle to segregate the genome within the nucleus while simultaneously nucleating cytoplasmic microtubules that orient the nucleus for delivery of a genome into each of the daughter cells.

 

How is the nuclear envelope remodeled? Our ability to study nuclear envelope-associated protein structures at high resolution using structured-illumination microscopy (SIM), iterative three-dimensional single particle averaging (SPA) and bimolecular fluorescence complementation (BiFC) have provided important mechanistic insight into this question.


 

INM transport

 

Mitotic strategies. Adapted from Smoyer and Jaspersen Trends Cell Biol. 2014


What sets the size of the nucleus and the spindle? Are these coupled during closed mitosis?

Faithful segregation of chromosomes involves many events, including duplication of microtubule-organizing centers, known as spindle pole bodies (SPBs) in fungi, once and only once per cell cycle. The microtubule-organizing center must also access DNA and grow to a sufficient size to nucleate enough microtubules for chromosome segregation.

 

We have a longstanding interest in the mechanism of SPB duplication and spindle formation. The minimal spindle found in yeast serves as an excellent model to understand the signaling cues that regulate SPB size and microtubule nucleation.

 

SPB duplication

 

Microtubule nucleation mechanism. Adapted from Cavanaugh and Jaspersen Annu. Rev. Genet. 2017.



How are nuclear components partitioned during cell division? How are nuclear pore complexes distributed on the nuclear membrane?

Nuclear pore complexes (NPCs) are located at multiple sites throughout the nuclear envelope to facilitate bidirectional movement of proteins and RNAs across the membrane. NPCs also have transport-independent functions, playing roles in chromosome organization, gene expression and nuclear integrity.

 

The number and distribution of NPCs is non-random, however, how NPC organization and function are controlled is poorly understood.  Using high resolution imaging, we can map NPC organization in live cells, allowing us to investigate NPC heterogeneity and study dynamic mechanisms that control NPC distribution.

 

Figure 6-FCCS-final

 

Visualization of nuclear envelope fenestra at fission yeast spindle poles (magenta) and nuclear pores (green) using SIM. (Andrew Bestul)