Localization and Behavior of Nuclear Lamin Proteins 
Dallin North 
Advisors: Dan Levy, Ph.D., Chase Wesley, B.S. 
May 14th, 2020 
University of Wyoming Department of Molecular Biology 
  
Abstract  
Nuclear size has been observed to be irregular in cancer cells, and as such can be used to 
phenotypically identify cancer cells and their stages. Many cancers have unusually large nuclei, 
and in certain cancers, nuclear size increases as the cancer becomes more aggressive. In order 
to fully understand the impact nuclear size has in cancer, and determine to what extent it is a 
cause or effect, we must understand how nuclear size is controlled. Although significant 
progress has been made over the past few years, many of the mechanisms and intricacies 
behind nuclear size regulation remain unknown. This project aims to determine one mechanism 
by which nuclear lamin proteins, which partially comprise the nucleoskeleton and help provide 
some structure to the organelle, help to control nuclear size. Previous research has suggested 
nuclear lamins may hold an important role in nuclear size regulation. Overall, this project 
intends to create an easily observable model system in human HeLa cells. This cell line, via 
CRISPR technology, will be genetically modified to endogenously tag Lamin A/C with enhanced 
green fluorescent protein (eGFP). This will allow Lamin A/C dynamics to be measured with 
Fluorescence Recovery After Photobleaching (FRAP) techniques. Additionally, as high protein 
kinase C (PKC) β activity has been implicated in the reduction of nuclear size in HeLa and MRC-5 
fibroblast cells, we plan to introduce a constitutively active version of PKC β to view the 
resulting behavior on dynamics. Ideally, these experiments will shed some light on how Lamin 
A/C dynamics relate to nuclear size. The results may be utilized with current cancer research to 
hopefully provide more understanding to the way cancer works or maybe even potential 
treatments. 
  
Introduction  
The nucleus is a common organelle present in most eukaryotic cells and is an integral part of 
cellular structure and function. It contains most of a cell’s genetic information in the form of 
concentrated DNA. This DNA is directly responsible for all the cell’s growth, behavior, and 
production via gene expression. Many steps towards the regulation of gene expression take 
place inside the nucleus: the central information hub of a cell. The nucleus consists of an outer, 
two-part membrane system known as the nuclear envelope, which is continuous with the 
endoplasmic reticulum. Inside the nucleus exists the nucleoplasm, in which the nucleolus (a 
small body involved in the synthesis of ribosomes), chromosomes, and other nuclear bodies 
remain suspended. Nuclear pores stretch across the envelope for regulation of transportation 
of large particles. A network of proteins known as the nuclear lamina form a meshwork on the 
inner surface of the nuclear membrane, functioning as a form of structural support and helping 
with protein anchoring. This mesh is composed of intermediate filaments called nuclear lamins. 
Lamins are synthesized outside in the cytoplasm and transported inside where they join the 
existing lamins. Current research suggests that nuclear lamins play a part in nuclear size 
regulation (1). Lamins exist in two forms, A type lamins (encoded by the LMNA gene) and B type 
lamins (encoded by the LMNB1 and LMNB2 genes). They are structurally similar, at the amino 
N-terminus, they start with a globular head domain, followed by an alpha-helical rod domain, 
with an immunoglobulin (Ig) domain at 
their C-terminus. Lamins also have a 
nuclear localization sequence located 
between the alpha-helical rod and 
Immunoglobulin domains, to direct the 
protein to its correct location in the 
nucleus. A-type lamins (generally present 
in differentiated cells) possess a neutral 
iso-electric point and an expanded C-
Figure 1: Lamin Polymerization and Formation of Intermediate 
Filaments (3). 
terminal tail, while the more universal B-
type lamins possess an acidic iso-electric point (2, 3). Lamins form the nuclear lamina via a 
process called polymerization. As shown in Figure 1, lamins dimerize, parallelly matching their 
globular head domains and their rod-like domains. Dimers then can form polymers, and then 
form protofilaments when bound in an opposite direction. Finally, the protofilaments can form 
the intermediate filaments, the basic unit of the nuclear lamina. DNA replication and repair, 
gene expression, chromatin organization, and signal transduction are all known to incorporate 
lamin interactions to some extent (3, 4). A significant amount of research has linked disruptions 
in the function of lamin proteins to abnormal nuclear morphologies. Previous studies in mouse 
models have demonstrated that embryonic fibroblasts with reduced lamin A/C resulted in a 
phenotypically decreased nuclear stiffness and deformed nuclei, while cells depleted of lamin 
B1 display irregular meshwork size and nuclear blebs (5-7). In Xenopus frogs, research has 
shown that alteration of the amount of specific lamin domains or specific posttranslational 
modifications to lamin proteins are sufficient to cause changes nuclear size. Nuclear expansion 
was impeded in Xenopus egg extract when the C-terminal Ig-fold motif of lamin B3 was added 
(8). Xenopus egg extract that was depleted of lamin B3 yielded small nuclei that failed to 
expand normally (9, 10). Further research indicated that Xenopus nuclear size was impacted by 
lamin expression levels in vivo (1). Finally, protein kinase C (PKC) activity has been implicated in 
reducing nuclear size in early Xenopus development, by phosphorylating lamin B3 (11). 
Research Aims and Protocol  
The aim of this research is to determine the mechanism underlying the reduction of nuclear size 
that occurs when Lamin A/C undergoes phosphorylation by PKC β. To begin, we will unfreeze 
and begin culture of a HeLa cell line. This will be followed by utilization of the CRISPR/cas9 gene 
editing system to insert a genetically modified fragment of the LMNA gene. This will result in 
cells with Lamin A/C endogenously tagged with eGFP, in order to measure its dynamics with 
FRAP techniques with and without the presence of constitutively active PKC isotypes. An 
endogenous Lamin A/C tag would allow for eGFP tagged Lamin A/C to be expressed at 
physiological levels, avoiding complications that that may occur with more traditional 
overexpression techniques. A FACS (fluorescence-activated cell sorting) machine would then be 
used to sort out the modified cells after transfection, create clonal populations of modified 
cells, and then use genetic techniques to confirm the genome modification occurred correctly 
without damaging other LMNA alleles. Finally, we will transfect the new cell line with 
constitutively active PKC constructs and use FRAP to determine how this will affect their Lamin 
A/C dynamics. 
  
Significance to Biomedical Field  
In cancer cells, it has been observed that the nucleus tends to become irregularly enlarged. The 
scaling ratio is often skewed in cancer cells and is thus used as an identification marker for 
cancer presence (12, 13). At this point, it is unknown to what extent inappropriate nuclear size 
is a cause or effect of cancer. If the mechanisms by which lamins influence nuclear size can be 
better understood, it could provide insight into a way in which nuclear size is mis-regulated in 
cancer. In addition to cancer relevance, lamin defects can result in laminopathies, genetic 
disorders caused by significant mutations in lamin genes. Laminopathies and similar nuclear 
envelopathies have been seen to cause dermo/neuropathy, muscular dystrophy, lipodystrophy, 
leukodystrophy, dysplasia, and progeria (14). Further knowledge of lamin behaviors in both 
cancer fields and laminopathy fields could eventually provide key information for a new 
direction in the treatment of these conditions or improve upon treatments already in use. 
Progress to Date 
To date, a base HeLa cell line was successfully started and split into separate testing flasks: Wild 
Type, 2x HDR eGFP-LMNA, 3x HDR eGFP-LMNA, and GFP-H2B overexpression (to serve as a 
positive fluorescence control). Groups denoted “2x” and “3x” were created and referenced to 
the different eGFP-LMNA repair construct proportions that would transfect cells, to attempt to 
see how construct concentration contributes to genome editing. Finally, an additional control 
was created which only received a GFP-Lamin A overexpression plasmid and no Cas9 enzyme. 
As shown in Figures 1 and 2, the transfection was successful and a small population of cells 
uptook the eGFP sequence into their LMNA gene, and successfully began expressing 
endogenously eGFP tagged lamin A/C. Although no formal measurements were made, both 
concentration samples yielded approximately less than 1% of modified cells. Unfortunately, 
access to a FACS machine was difficult. Over the course of 2 months, the eGFP+ cells were lost 
before a reliable method of sorting GFP positive cells from the rest of the population could be 
procured. Once undergraduate research is again allowed, we will need to transfect another 
HeLa cell line, then sort immediately to create clonal populations. At this point, University of 
Wyoming policy has undergraduate research put on 
hold until coronavirus restrictions ease. As such, this 
experiment has been put on pause for the time being. 
Discussion 
The success in getting even temporary eGFP+ cells 
proved that the proposed genome edit is possible, and 
that the creation of clonal populations of edited HeLa Figure 2: Successful Genome Edited HeLa Cells 
Transfected eGFP+ HeLa cells are shown in the 
circled area, as shown in the TRITC channel 
cells is feasible. However, immediate access to a FACS (displaying lamin A/C). Un-transfected cells are 
shown outside the circled area.  
machine will be required when the transfection is 
repeated. It is unknown why the eGFP+ cells eventually 
disappeared from the sample populations, but in the 
future, immediate cell isolation and propagation can 
solve this problem.  
There are likely several factors responsible for the low 
efficiency of the CRISPR-based genome editing. In Figure 3: Successful Genome Edited HeLa Cells 
Transfected eGFP+ HeLa cells fluorescing under 
general, high transfection yields have proven the microscope, as shown in the FITC channel 
(showing expression of eGFP). Un-transfected 
cells are not visible. 
themselves difficult to achieve and success of genome editing can vary based on target gene 
sequence. Specifically, the targeted region of the LMNA gene had limited PAM (protospacer 
adjacent motif) sites which are required for Cas9 nuclease activity. As noted above, the 
transfection efficiency was not high enough for all sample cells to receive the eGFP construct. 
To repair double stranded DNA breaks, the cell naturally employs the NHEJ (non-homologous 
end joining) mechanism to a greater extent than repair through HDR (Homology Directed 
Repair). Only HDR allows for this form of genome editing (15). To improve transfection efficacy, 
increasing construct concentration may lead to increased chances for construct uptake. To 
compensate for the lack of Cas9 binding sites in LMNA, it could be worth testing larger gRNA 
sequences that may have access to more PAM sites. However, as a rule of thumb, larger 
constructs generally lead to more problems. 
In conclusion, the completion of this project will result in the culmination of a testing platform 
designed to observe and test the effects of PKC β phosphorylation on lamin A/C, and the 
changes in lamina interactions that follow. Ideally, these experiments will also clarify how lamin 
A/C dynamics relate to nuclear size, and further our knowledge of cancer progression in order 
to potentially draft new treatments. 
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