PROJECT ABSTRACT The nuclear envelope encloses the nucleus and establishes a critical protective barrier around genomic DNA. Proteins embedded in and associated with the two membrane bilayers of the nuclear envelope play crucial roles in mediating chromatin organization, nuclear positioning, and connections to the cytoskeletal network. There is growing appreciation that disruption of nuclear envelope integrity is linked to multiple aging-associated diseases and contributes to genomic instability in cancer. Upon each cell division in mammalian cells, the nucleus is dismantled and reformed. Scrutiny of nuclear envelope assembly has revealed the remarkable feature that two distinct domains are present at the nascent nuclear envelope. Core regions at the center of each face of the anaphase chromatin disk are a site of microtubule remodeling, and non-core regions at the disk periphery are a site of rapid nuclear pore formation. This specialization facilitates quick, coordinated enclosure of chromatin. Progression to interphase nuclear architecture involves elimination of the distinction between these regions and further expansion of membrane. Completion of nuclear assembly, however, does not mean that the nuclear envelope is static. Indeed, emerging evidence supports the notion that continuous adaptations must take place at the nuclear envelope to maintain nuclear envelope structure and, in some cases, to sculpt new architectural features. Using approaches ranging from timelapse microscopy to proximity labeling, this proposal seeks to address fundamental questions about how the nuclear envelope is formed and how its organization and topology are maintained. Toward this end, the first aim is to determine molecular pathways that underlie dynamic organization of the nascent nuclear envelope. This aim will focus on testing roles of the inner nuclear membrane protein LEM2, defining motifs that direct core/non-core regional targeting, and investigating how dissolution of the core region is accomplished. Added insight will be gained by examining how the nuclear envelope forms at micronuclei, which arise from mis-segregated chromosomes; in particular, we will investigate how a fragile nuclear envelope forms in a subpopulation of micronuclei. The second aim is to elucidate the role of LEM2 in dynamic events at the interphase nuclear envelope and learn how these are altered in disease-associated alleles of LEM2. This aim takes advantage of a system in which nuclear membrane deformation can be induced to test mechanisms that contribute to the resilience of nuclear envelope architecture. Results reported in the literature, as well as our observations, implicate LEM2 in responding to these perturbations. These complementary aims will yield a deeper understanding of nuclear envelope formation and reveal pathways that play critical roles in nuclear architecture, as well as probe how they go awry at micronuclei and are disrupted in human disease.