International review of cell and molecular biology, volume 322

VOLUME THREE HUNDRED AND TWENTY TWO

INTERNATIONAL REVIEW OF
CELL AND MOLECULAR
BIOLOGY


International Review of Cell
and Molecular Biology
Series Editors
GEOFFREY H. BOURNE
JAMES F. DANIELLI
KWANG W. JEON
MARTIN FRIEDLANDER
JONATHAN JARVIK

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ROBERT A. BLOODGOOD
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MICHAEL MELKONIAN
KEITH E. MOSTOV
ANDREAS OKSCHE
MADDY PARSONS
TERUO SHIMMEN
ALEXEY TOMILIN
GARY M. WESSEL


VOLUME THREE HUNDRED AND TWENTY TWO

INTERNATIONAL REVIEW OF
CELL AND MOLECULAR
BIOLOGY

Edited by

KWANG W. JEON

Department of Biochemistry
University of Tennessee
Knoxville, Tennessee

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CONTRIBUTORS
Fiorenza Accordi
Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome,
Italy
Jaap D. van Buul
Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory,
Academic Medical Center, University of Amsterdam, The Netherlands
Claudio Chimenti
Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome,
Italy
Annalena Civinini
Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome,
Italy
Enrico Crivellato
Department of Experimental and Clinical Medicine, Section of Anatomy, University of Udine,
Italy
Anna E. Daniel
Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory,
Academic Medical Center, University of Amsterdam, The Netherlands
Lisa J. Edens
Department of Molecular Biology, University of Wyoming, Laramie, WY, United States
of America
Valentina P. Gallo
Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Italy
Stephanie L. Gupton
Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC,

United States of America; Neuroscience Center and Curriculum in Neurobiology, University
of North Carolina, Chapel Hill, NC, United States of America; Lineberger Comprehensive
Cancer Center, University of North Carolina, Chapel Hill, NC, United States of America
Predrag Jevtic´
Department of Molecular Biology, University of Wyoming, Laramie, WY, United States of
America

ix


x

Contributors

Eric J. Kremer
Institut de Ge´ne´tique Mole´culaire de Montpellier, Universite´ de Montpellier, Montpellier,
France
Jeffrey Kroon
Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory,
Academic Medical Center, University of Amsterdam, The Netherlands
Daniel L. Levy
Department of Molecular Biology, University of Wyoming, Laramie, WY, United States of
America
Fabien Loustalot
Institut de Ge´ne´tique Mole´culaire de Montpellier, Universite´ de Montpellier, Montpellier,
France
Shalini Menon
Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC,
United States of America
Francisco Rivero

Hull York Medical School, University of Hull, Hull, United Kingdom
Sara Salinas
Institut de Ge´ne´tique Mole´culaire de Montpellier, Universite´ de Montpellier, Montpellier,
France
Ilse Timmerman
Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory,
Academic Medical Center, University of Amsterdam, The Netherlands
Lidija D. Vukovic´
Department of Molecular Biology, University of Wyoming, Laramie, WY, United States of
America
Cortney Chelise Winkle
Neuroscience Center and Curriculum in Neurobiology, University of North Carolina,
Chapel Hill, NC, United States of America
Huajiang Xiong
Hull York Medical School, University of Hull, Hull, United Kingdom


CHAPTER ONE

New Insights into Mechanisms
and Functions of Nuclear Size
Regulation
Lidija D. Vuković, Predrag Jevtić, Lisa J. Edens, Daniel L. Levy*
Department of Molecular Biology, University of Wyoming, [18_TD$IF]Laramie, WY, United States of America

*Corresponding author. E-mail:

Contents
1. Introduction
2. Overview of Cellular Structures and Activities that Contribute to Nuclear Size

Determination
2.1 Nuclear Structure and Models of Organelle Size Control
2.2 Genome Size and Ploidy
2.3 Chromatin State
2.4 Cell Size and Nucleocytoplasmic Ratio
2.5 Nucleocytoplasmic Transport
2.6 Intranuclear Structures
2.7 Extranuclear Structures
2.8 Cell-Cycle Effects
2.9 Signaling Pathways
3. Model Systems to Elucidate Mechanisms of Nuclear Size Regulation
3.1 Tetrahymena thermophila
3.2 Yeasts and Fungi
3.3 Plants
3.4 Caenorhabditis elegans
3.5 Drosophila melanogaster
3.6 Zebrafish
3.7 Xenopus
3.8 Mammalian Model Systems
4. Functional Significance of Nuclear Size and Morphology
4.1 Chromosome Positioning, Chromatin Organization, and Gene Expression
4.2 Nuclear Mechanics and Cell Migration
4.3 Nuclear Size and Morphology Changes in Cancer
4.4 Nuclear Envelopathies

International Review of Cell and Molecular Biology, Volume 322
ISSN 1937-6448
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© 2016 Elsevier Inc.
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5. Conclusions
Acknowledgments
References

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Abstract
Nuclear size is generally maintained within a defined range in a given cell type.
Changes in cell size that occur during cell growth, development, and differentiation
are accompanied by dynamic nuclear size adjustments in order to establish appropriate nuclear-to-cytoplasmic volume relationships. It has long been recognized that
aberrations in nuclear size are associated with certain disease states, most notably
cancer. Nuclear size and morphology must impact nuclear and cellular functions.
Understanding these functional implications requires an understanding of the
mechanisms that control nuclear size. In this review, we first provide a general
overview of the diverse cellular structures and activities that contribute to nuclear
size control, including structural components of the nucleus, effects of DNA amount
and chromatin compaction, signaling[20_TD$IF] and transport pathways that impinge on the
nucleus, extranuclear structures, and cell cycle state. We then detail some of the key
mechanistic findings about nuclear size regulation that have been gleaned from a
variety of model organisms. Lastly, we review studies that have implicated nuclear size
in the regulation of cell and nuclear function and speculate on the potential functional
significance of nuclear size in chromatin organization, gene expression, nuclear
mechanics, and disease. With many fundamental cell biological questions remaining
to be answered, the field of nuclear size regulation is still wide open.

1. INTRODUCTION
Cell and nuclear sizes differ greatly among different species, as well as
within the same organism when comparing different cell types. Even in the
same tissue, cell and nuclear sizes can vary depending on the developmental
stage, state of cell differentiation, a variety of external factors, and cellular
transformation. How nuclear size and shape affect cell physiology is still
unclear, but it is certainly possible that nuclear morphology impacts chromatin organization and gene expression. Elucidating the functional significance of nuclear size necessitates an understanding of the mechanisms that
control nuclear size. This is particularly important in the case of pathologies
in which nuclear morphology is altered, most notably cancer, where it is
unclear if changes in nuclear size and shape are a cause or consequence of
disease.
In this review, we first provide a general overview of the diverse cellular
structures and activities that are relevant to the regulation of nuclear size,


New Insights into Mechanisms and Functions of Nuclear Size Regulation

3

including ploidy, chromatin condensation, the nucleocytoplasmic ratio,
and nuclear transport (Fig[21_TD$IF]. 1). We next turn to different model systems that
have and will continue to shed light on mechanisms of nuclear size regulation
(Fig[21_TD$IF]. 2). We review how nuclear size is regulated by soluble transport factors,
structural components of the nuclear envelope [2_TD$IF](NE), signaling pathways, and
extranuclear structures like the endoplasmic reticulum and cytoskeleton.
Lastly, we discuss demonstrated or proposed roles for nuclear size in chromosome organization, gene expression, nuclear mechanics, and pathology.
In the last decade, the great complexity of nuclear structure and function has
begun to emerge, and here we provide a broad overview of how the regulation of nuclear morphology contributes to this complexity.

2. OVERVIEW OF CELLULAR STRUCTURES AND
ACTIVITIES THAT CONTRIBUTE TO NUCLEAR SIZE
DETERMINATION
Organelle size and morphology must have important implications for
organelle and cellular function (Edens et al., 2013; Heald and Cohen-Fix,
2014; Jevtic et al., 2014). Understanding these functional implications first
requires elucidation of the mechanisms that control organelle size. When it
comes to nuclear size regulation, a number of potential mechanisms have
been implicated including structural components of the nucleus, effects of
DNA amount and chromatin compaction, signaling and transport pathways
that impinge on the nucleus, extranuclear structures, and cell cycle state. In
this section, we provide a general overview of these diverse mechanisms that
contribute to nuclear size control. Each subsection deals with a general
theme relevant to the regulation of nuclear size. At the end of each subsection, we refer to later sections in the review that delve into greater detail with
respect to that mechanism.

2.1 Nuclear [23_TD$IF]Structure and Models of Organelle Size Control
The NE is composed of a double lipid bilayer. The outer nuclear membrane
(ONM) is a continuous extension of the endoplasmic reticulum (ER). In
metazoan nuclei, the inner nuclear membrane (INM) is lined on its nucleoplasmic face by the nuclear lamina, a meshwork of intermediate lamin
filaments. The INM and ONM are fused at sites of nuclear pore complex
(NPC) insertion. The NPC mediates nucleocytoplasmic transport of proteins and mRNA. [24_TD$IF]Linker of nucleoskeleton and cytoskeleton (LINC)


4

[(Figure_1)TD$IG]

Lidija D. Vuković et al.

New Insights into Mechanisms and Functions of Nuclear Size Regulation

Figure 1 [1_TD$IF]Nuclear structure and chapter overview. [2_TD$IF]On the left side of the diagram, key nuclear structures are depicted. The right side of the
diagram shows an overview of the three main sections of the chapter, including illustrative images from some of the studies reviewed. For
section [3_TD$IF]2, the ploidy example demonstrates that a 16-fold increase in ploidy (bottommost cell) in S. pombe does not affect nuclear size. [4_TD$IF][Image
was adapted from (Neumann and Nurse, 2007), made available by creativecommons.org/licenses/by-nc-sa[5_TD$IF]/3.0/.] The cell cycle example shows
that when REEP3/4 are knocked down, membrane fails to be cleared from metaphase chromosomes, leading to nuclear morphology defects in
the subsequent interphase. [4_TD$IF][Image was used with permission from (Schlaitz et al., 2013[6_TD$IF])] The signaling image shows that in Xenopus, nuclear
cPKC levels are low in early development (a) and high later in development (b), correlating with reductions in nuclear size. [4_TD$IF][Image was adapted
from (Edens and Levy, 2014a), made available by creativecommons.org/licenses/by-nc-sa[7_TD$IF]/3.0/.] For section 3, the budding yeast example shows
electron microscopy images of two different size wild-type G1 cells generated by varying the growth conditions. Nuclei are outlined. Scaling of
nuclear size with cell size is evident. [4_TD$IF][Image was used with permission from (Jorgensen et al., 2007[6_TD$IF])] In the Xenopus example, cell and nuclear
sizes become smaller during progression from early to later stages of development. [4_TD$IF][Image was used with permission from (Jevtic and Levy,
2015[6_TD$IF])] In the mammalian example, U2OS tissue culture cells overexpressing the ER-tubule shaping protein Rtn4 exhibit reduced nuclear
expansion rates and smaller nuclei. [8_TD$IF][The image was adapted from (Anderson and Hetzer, 2008), made available by creativecommons.org/
licenses/by-nc-sa[9_TD$IF]/3.0/.] For section 4, the laminopathy example demonstrates that nuclear morphology is highly disrupted in HGPS patients
(bottom nucleus) (Scaffidi et al., 2005), made available by creativecommons.org/licenses/by/4.0/. The cancer example shows our unpublished
data in which nuclei are enlarged in metastatic melanoma cells compared to normal melanocytes. In the cell migration example, HT1080
fibrosarcoma cells are shown migrating through microfluidic channels of different dimensions. The shape of the nucleus must change to pass
through narrow channels. [8_TD$IF][The image was used with permission from (Denais and Lammerding, 2014[10_TD$IF])]

5


[(Figure_2)TD$IG]

Figure 2 Model organisms and factors that control nuclear size. [2_TD$IF]The left column shows
model organisms that have been used to elucidate mechanisms of nuclear size regulation.
The other columns list how manipulating specific proteins and structures affects nuclear

size and morphology. The relevant references are included throughout the text.


New Insights into Mechanisms and Functions of Nuclear Size Regulation

7

complexes span the NE establishing connections between the cytoskeleton
and nuclear interior (Rothballer and Kutay, 2012a,[25_TD$IF]b; Simon and Wilson,
2011; Wilson and Berk, 2010). The usual shape of the nucleus is spherical or
ellipsoid (Walters et al., 2012), and in some cases extensions of the NE reach
into the interior of the nucleus in the form of a nucleoplasmic reticulum
(Malhas et al., 2011) (Fig[21_TD$IF]. 1).
A number of different models for organelle size control have been proposed, and many of these may be relevant to the nucleus (Chan and Marshall,
2012; Marshall, [26_TD$IF]2002, 2012). One model is that the sizes of individual
components of the structure act as rulers to dictate the overall size of the
structure (Tskhovrebova and Trinick, 2012). Such a ruler mechanism has
been demonstrated for measuring the length of cilia and flagella
(Oda et al., 2014), muscle sarcomere thin filaments (Fernandes and
Schock, 2014), chromosomes (Neurohr et al., 2011), and even RNA
(McCloskey et al., 2012). Another model for organelle size control is one
in which fixed amounts of organellar building blocks determine its ultimate
size (Goehring and Hyman, 2012). These limiting component models are
relevant to size control of the mitotic spindle (Good et al., 2013; Hazel et al.,
2013) and Golgi (Ferraro et al., 2014; Romero et al., 2013). Regulated
synthesis of organelle structural components can also control size, as in the
case of lipid droplets (Wilfling et al., 2013).
More dynamic mechanisms for organelle size control have also been
proposed, for instance[27_TD$IF], balancing rates of organelle assembly and disassembly
that determine steady-state size. Examples of size control that fit this model

include flagella (Ludington et al., 2012) and peroxisomes (Mukherji and
O’Shea, 2014), and likely most membrane-bound organelles. In [28_TD$IF]case of
the nucleus, invoking several of these models may be necessary to fully
account for nuclear size regulation, and we will touch on these models
throughout the review. Also see Sections [29_TD$IF]3.2–3.5, 3.7, 3.8, and 4.1–4.4.

2.2 Genome Size and Ploidy
The correlation between genome and nuclear size has been known for over a
century (Gregory, 2001, 2011; Umen, 2005). It is therefore tempting to
speculate that nuclear size is determined by the amount of nuclear DNA.
However, abundant phenomenological and experimental evidence demonstrates that other factors must contribute to nuclear size. Different cell types
within the same species exhibit nuclear size differences, despite having the
same genome content, and nuclear size varies during early development


8

Lidija D. Vuković et al.

while the DNA amount per cell remains constant (Altman and Katz, 1976;
Butler et al., 2009; Faro-Trindade and Cook, 2006; Oh et al., 2005;
Thomson et al., 1998). Experimental manipulation of DNA content often
has a minimal impact on nuclear size. In fission yeast, a 16-fold increase in
nuclear DNA amount did not affect nuclear size (Neumann and Nurse,
2007) (Fig[21_TD$IF]. 1). Furthermore, an abrupt increase in nuclear size was not
observed at the time of DNA replication as might be expected if DNA
amount significantly impacted nuclear size (Jorgensen et al., 2007;
Neumann and Nurse, 2007). Consistent with these results, nuclei in mammalian tissue culture cells expanded normally during interphase even when
DNA replication was blocked (Maeshima et al., 2010).
Nonetheless, ploidy changes can have important implications for cellular

function. Programmed polyploidization in mammalian cells is an adaptive
response to stress and injury (Pandit et al., 2013). In Drosophila melanogaster,
polyploidization of glia is necessary to maintain integrity of the blood-brain
barrier (Unhavaithaya and Orr-Weaver, 2012) and plays a role in wound healing
in the adult epithelium (Losick et al., 2013). In different diatom species, genome
size correlates with cell division rates (Sharpe et al., 2012), and altered ploidy in
salamanders impacts cell and animal size (Fankhauser, 1939, 1945a, b). It is
unknown whether these functional effects might be mediated through changes
in nuclear size. Also see Sections 3.1, 3.2, 3.5, 3.7[207_TD$IF], and 4.3.

2.3 Chromatin [208_TD$IF]State
In addition to DNA amount, chromatin compaction is another feature that
potentially impacts nuclear size and morphology. The large number of
proteins known to interact with and modify chromatin complicates this
question (Kustatscher et al., 2014), although roles for condensins and histones have emerged. For example, increasing condensin II-mediated chromatin compaction in Drosophila caused distortion of NE morphology
(Buster et al., 2013). An analysis of 160 eukaryotic genomes showed that
as genome size increased during evolution, the amino terminus of histone
H2A has acquired arginine residues that confer increased chromatin compaction. Addition of arginine residues to the yeast H2A resulted in increased
chromatin compaction and reduced nuclear volume, while mutating arginine residues in human H2A led to chromatin decompaction and increased
nuclear size (Macadangdang et al., 2014).
It is worth [31_TD$IF]noting that chromatin compaction might also indirectly
impact nuclear size. Yeast cells increase compaction of long chromosome


New Insights into Mechanisms and Functions of Nuclear Size Regulation

9

arms during mitosis to ensure complete chromosome segregation
(Neurohr et al., 2011), whereas Drosophila cells transiently elongate during

anaphase (Kotadia et al., 2012). In both instances, this might affect nuclear
size in the subsequent interphase. Histone H3 methylation status has been
shown to dictate chromatin regions that associate with the nuclear lamina, so
called lamina-associated domains (LADs) (Harr et al., 2015), and certain long
noncoding RNAs regulate histone methylation (Wang et al., 2011b).
Chromatin organization might, in turn, affect nuclear size. Also see
Sections 3.1, 3.2, 3.4, 3.7, 3.8, 4.1[32_TD$IF], and 4.3.

2.4 Cell Size and Nucleocytoplasmic Ratio
It has long been recognized that the nuclear-to-cytoplasmic (N/C) volume
ratio is maintained at a roughly constant value in normal cells (Wilson, 1925),
and this ratio is often perturbed in cancer cells (Chen et al., 2010; Hokamp
and Grundmann, 1983; Zhuang et al., 2008). Classic experiments showed
that nuclear size is dynamically sensitive to cytoplasmic volume. When hen
erythrocytes were fused with HeLa cells, the erythrocyte nuclei grew larger
and became transcriptional active (Harris, 1967). Somatic nuclei injected
into Xenopus eggs or oocytes also expanded, with nuclei exposed to larger
cytoplasmic volumes enlarging more (Gurdon, 1976; Merriam, 1969).
Manipulating cytoplasmic partitioning in sea snail embryos demonstrated
that nuclei within larger cytoplasmic volumes were larger than nuclei within
small cytoplasmic volumes (Conklin, 1912). Yeast studies have also shown
that nuclear size is sensitive to cytoplasmic volume (Jorgensen et al., 2007;
Neumann and Nurse, 2007). The underlying mechanisms responsible for
sensing and regulating the N/C ratio are not yet fully understood.
An equally important question in the context of the N/C ratio is how cell
size is controlled. In principle, the two relevant parameters are cell growth rate
and cell division rate. What are the mechanisms responsible for sensing cell size
(Umen, 2005)? In fission yeast, two models have emerged. By one model, a
gradient of Pom1, a cell polarity kinase located at the cell ends, acts as a sensor
of cell size. As cells elongate, Pom1 levels decrease at the center of the cell and

upon reaching a critical low level, mitosis is induced (Martin and BerthelotGrosjean, 2009; Moseley et al., 2009). In another model, total cell surface area
is sensed by Cdr2, a peripheral membrane kinase (Pan et al., 2014). In budding
yeast, accumulation of G1 cyclins appears to act as a proxy for cell size (Cross,
1988; Nash et al., 1988; Zapata et al., 2014). As in budding yeast, cyclin
expression controls the number of times erythroid precursors divide during


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Lidija D. Vuković et al.

differentiation, influencing erythrocyte size and number (Sankaran et al., 2012).
The situation in mammalian cells is likely more complex than in yeasts (Echave
et al., 2007; Kafri et al., 2013; Tzur et al., 2009).
Cell size certainly has important implications for cell function that might
influence nuclear size. For instance, RNA and protein synthesis tend to scale
with cell size (Kempe et al., 2015; Marguerat and Bahler, 2012; Sato et al.,
1994; Schmidt and Schibler, 1995). Neuron length sensing depends both on
transcription factors levels and the action of cytoskeletal motor proteins
(Albus et al., 2013). Wnt signaling concomitantly increases cellular protein
content and cell size (Acebron et al., 2014). A genetic screen in Jurkat cells
identified Largen as a protein that increases cell size through increased
expression of mitochondrial proteins (Yamamoto et al., 2014). Conversely,
experimentally increasing cell size in mouse hepatocytes led to a reduction in
mitochondrial gene expression (Miettinen et al., 2014).
Cell size and rates of cell division are also coupled to developmental
morphogen gradients, environmental signals, and growth conditions
(Averbukh et al., 2014; Slavov and Botstein, 2013). On the other hand,
cytokinesis rates appear to be independent of cell size, due to the fact that
larger contractile rings constrict faster than smaller ones (Calvert et al., 2011;

Carvalho et al., 2009; Turlier et al., 2014). Active spindle positioning and
plasma membrane expansion mechanisms ensure that cell division occurs in
the middle of the cell to generate daughter cells of equal size (Kiyomitsu and
Cheeseman, 2013). Cell shape control is also critical for cell function. Failure
for cells to round at mitosis causes defects in spindle assembly and mitotic
progression (Cadart et al., 2014; Lancaster et al., 2013; Ramanathan et al.,
2015), and the ability of cells to switch between discrete cell shapes appears to
be under genetic control (Yin et al., 2013). Open questions remain about
how cell size and shape impinge on the control of nuclear morphology. Also
see Sections [3_TD$IF]3.2–3.5, 3.7, 3.8, and 4.3.

2.5 Nucleocytoplasmic [34_TD$IF]Transport
One mechanism that contributes to the regulation of nuclear size in a variety
of systems is nucleocytoplasmic transport. Classical nuclear import of proteins
containing a nuclear localization signal (NLS) is mediated by karyopherins
of the importin α/β families. Expression of different importin isoforms
controls nuclear import of specific cargo molecules. Nuclear import begins
when NLS cargos interact either directly with importin β or indirectly
through association with an importin α adapter. Importin β interacts with


New Insights into Mechanisms and Functions of Nuclear Size Regulation

11

nucleoporins (Nups) of the NPC that contain phenylalanine/glycine (FG)
repeats. Once within the nucleus, the importin/cargo complex dissociates
upon binding of Ran-GTP to importin β, releasing the cargo in the
nucleus. Ran is a small monomeric GTPase critical to [39_TD$IF] directional nucleocytoplasmic transport. Intranuclear Ran is predominantly GTP bound
because the Ran guanine nucleotide exchange factor (RanGEF), RCC1,

is bound to the chromatin. In nuclear export, proteins containing a nuclear
export signal (NES) complex with an exportin, such as CRM1, and RanGTP for directed transport to the cytoplasm through the NPC
(Xu et al., 2012). The Ran GTPase activating protein (RanGAP) is localized to the cytoplasmic surface of the NPC, so upon export Ran’s bound
nucleotide is hydrolyzed to GDP and the exported cargo is released in the
cytoplasm (Alberts et al., 1994).
Dedicated transport factors play important roles in regulating the nucleocytoplasmic distribution of the importins and Ran. After a round of nuclear
import, CAS is responsible for recycling importin α back to the cytoplasm for
additional cycles of import. NTF2 is a protein that largely associates with the
NPC and is responsible for recycling of cytoplasmic Ran-GDP back into the
nucleus, where is it converted to Ran-GTP by RCC1 (Bayliss et al., 1999;
Clarkson et al., 1997; Morrison et al., 2003; Smith et al., 1998). Kinetics of
nuclear import based on cargo size and competition may have important
implications for the regulation of nuclear size (Feldherr et al., 1998; Hodel
et al., 2006; Hu and Jans, 1999; Lane et al., 2000; Lyman et al., 2002; Mincer and
Simon, 2011; Timney et al., 2006). It is worth [31_TD$IF]noting that many nuclear
proteins do not possess a canonical NLS, and importin-independent pathways
have recently been elucidated (Lu et al., 2014). Similarly, some proteins of the
INM reach the interior of the nucleus through importin-independent pathways
(Boni et al., 2015; Katta et al., 2013; Ungricht et al., 2015), and large ribonucleoprotein complexes are able to leave the nucleus by budding of the NE,
bypassing the NPC entirely (Speese et al., 2012). Redistribution of nuclear
transport factors that occurs during differentiation and stress could contribute to
concomitant changes in nuclear size (Andrade et al., 2003; Huang and Hopper,
2014; Kose et al., 2012; Lieu et al., 2014; Rother et al., 2011; Whiley et al.,
2012; Yasuda et al., 2012). Also see Sections [40_TD$IF]3.1–3.4, 3.7, and 4.3.

2.6 Intranuclear [41_TD$IF]Structures
Related to nuclear size is the question of how the sizes of intranuclear
structures are determined and whether nuclear size impacts intranuclear

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Lidija D. Vuković et al.

structure. A variety of different membraneless RNA/protein bodies are
found within the nucleus, such as nucleoli, speckles, and Cajal, histone
locus, and PML bodies. Surprisingly, many of these structures have been
shown to exhibit fluid-like properties, with important implications for the
number and size distribution of these bodies within the nucleus
(Brangwynne, 2013; Toretsky and Wright, 2014). For example, nucleoli
within the large germinal vesicle (GV) of Xenopus oocytes can fuse
(Brangwynne et al., 2011). A network of actin filaments acts to keep
individual nucleoli separated, and disruption of actin causes nucleoli to
settle to the bottom of the nucleus under the force of gravity where they
fuse into one large nucleolus (Feric and Brangwynne, 2013). Clearly this is
an extreme example as the GV is on the order of 0.5 mm in diameter, but it
demonstrates that nuclear size has important implications for intranuclear
structure and organization. Mechanisms that regulate the assembly of such
ribonucleoprotein bodies are beginning to be elucidated (Nott et al.,
2015). Also see [42_TD$IF]Sections 3.2 and 3.4.

2.7 Extranuclear [41_TD$IF]Structures
The endoplasmic reticulum (ER) has also been shown to influence nuclear
size. The ER is an interconnected network of lipid bilayer membrane sheets
and tubules that is continuous with the NE. It has been proposed that there is
a tug-of-war relationship between the NE and ER membrane systems.
Altering the relative proportions of ER tubules and sheets can concomitantly
affect nuclear size (Anderson and Hetzer, 2007, 2008; Webster et al., 2009).
Proteins in the reticulon (Rtn) and REEP families shape ER membranes into
tubules and also stabilize membrane curvature at the edges of ER sheets

(Friedman and Voeltz, 2011; Shibata et al., 2010; Voeltz et al., 2006; West
et al., 2011).
Disruption of the tubular ER network inhibits nuclear expansion in
Xenopus egg extracts (Anderson and Hetzer, 2007), and ectopic expression
of Rtn4 alters nuclear size in Xenopus embryos (Jevtic and Levy, 2015).
Reticulon knockdown in U2OS osteosarcoma cells reduced ER tubule
formation, increased the amount of ER sheets, and accelerated NE formation leading to increased nuclear size. Conversely, reticulon overexpression
increased ER tubulation, reduced ER sheet formation, inhibited nuclear
expansion, and decreased nuclear size (Anderson and Hetzer, 2008) (Fig[21_TD$IF]. 1).
These experiments support the idea that the relative proportion of ER sheets
and tubules contributes to the regulation of NE growth and steady-state


New Insights into Mechanisms and Functions of Nuclear Size Regulation

13

nuclear size. Future experiments will address if other morphological proteins
of the ER also impact nuclear size, such as CLIMP63 that dictates ER sheet
spacing (Shibata et al., 2010), atlastins that mediate fusion of ER tubules into
three-way junctions (Hu et al., 2009; Orso et al., 2009), and Lunapark that
regulates three-way junction dynamics (Chen et al., [43_TD$IF]2012b, 2015a).
The microtubule and actin cytoskeletons also impact nuclear size and
morphology. One intriguing example involves cytoplasmic streaming, a
form of intracellular transport found in plants that is generated by the
movement of organelles, including the nucleus, along actin filaments under
the action of myosin XI. The exact functional role of this streaming is largely
unknown. High-speed and low-speed versions of [4_TD$IF]Arabidopsisthaliana myosin
XI were generated by varying the motor domain. Expression of high-speed
chimeric myosin accelerated cytoplasmic steaming and led to increased cell

and plant size. Conversely, low-speed myosin slowed cytoplasmic streaming
and cells and plants were smaller (Tominaga et al., 2013). Because there is a
relationship between cell and nuclear size, we speculate that the kinetics of
cytoplasmic streaming might influence nuclear size, although this was not
explicitly examined in this study. Also see Sections [45_TD$IF]3.2–3.8and 4.2.

2.8 Cell-Cycle Effects
The kinetics of some cell cycle events can have important implications for
nuclear size and morphology. During the metazoan cell cycle, nuclear envelope breakdown (NEBD) occurs prior to mitosis. This is in contrast to the
closed mitoses of many yeasts in which the nucleus remains intact during
mitosis (Sazer et al., 2014). NEBD is initiated by a variety of different kinases
including cyclin-dependent kinases (Cdks), protein kinase C (PKC), and
NIMA-related kinases. Key nuclear substrates in NEBD are lamins and
nucleoporins (Laurell et al., 2011; Mall et al., 2012). Interestingly, this
process seems to have been adapted during lens epithelial cell differentiation
to remove nuclei entirely from these cells (Chaffee et al., 2014). NEBD in
starfish oocytes is driven by an F-actin shell that fragments the NE membrane
(Mori et al., 2014), while an in vitro Xenopus assay for NEBD implicated the
microtubule cytoskeleton and Ran in NE rupture (Muhlhausser and Kutay,
2007). LINC complex proteins that connect the nucleus to the cytoskeleton
are also important in NEBD, and impairing NEBD can lead to mitotic
defects (Turgay et al., 2014).
After NEBD, mitotic spindle assembly and chromosome segregation
ensue. Components of the NE are important for this process, for example


14

Lidija D. Vuković et al.

lamins contribute to formation of a spindle matrix (Johansen et al., 2011;
Tsai et al., 2006). Interestingly, nuclear and cytoplasmic proteins do not
appear to mix homogeneously after NEBD, and this may have important
implications for spindle assembly and the subsequent cell cycle (Pawar
et al., 2014). In radial glial progenitor cells, dynein-dependent nuclear
migration during the G1 phase of the cell cycle is necessary for entry into
mitosis (Hu et al., 2013). Aside from the nucleus, other organelles must also
be faithfully segregated between the two daughter cells during mitosis
(Jongsma et al., 2015).
After anaphase, NE assembly occurs around the segregated daughter
chromosomes through targeting of ER tubules to the chromatin and spreading of membrane across the chromosomes (Anderson and Hetzer, 2007;
Clever et al., 2013; Schooley et al., 2012). It was recently demonstrated that
components of the ESCRT machinery are responsible for the membrane
fusion that gives rise to an intact NE (Olmos et al., 2015; Vietri et al., 2015).
Distinct mechanisms are responsible for NPC assembly during NE formation and for the insertion of NPCs into the intact NE during interphase
(D’Angelo et al., 2006; Doucet et al., 2010; Talamas and Hetzer, 2011).
Nuclear expansion during interphase occurs through a nuclear importdependent process. In mammalian tissue culture cells, the number of
NPCs and nuclear volume were observed to double during interphase,
and Cdk activity was involved in interphase NPC formation. Interestingly,
Cdk inhibition disturbed new NPC assembly but did not block nuclear
expansion, suggesting that NPC doubling during interphase is not required
for normal nuclear growth (Maeshima et al., [47_TD$IF]2010, 2011).
Proper NE assembly depends on the removal of microtubules and membrane from chromosomes. REEP3/4 are ER proteins required to clear
membranes from metaphase chromosomes through a microtubule-dependent process. Failure of this process leads to defective NE architecture, the
formation of intranuclear membrane structures, and defects in chromosome
segregation and the separation of daughter nuclei (Schlaitz et al., 2013) (Fig[21_TD$IF].
1). BAF is a chromatin-bound protein key to NE assembly that is usually
removed from the chromatin prior to mitosis. Mutations causing constitutive
chromatin association of BAF led to highly aberrant nuclear morphology,
likely resulting from a failure to clear membrane from the mitotic chromosomes (Molitor and Traktman, 2014). While microtubules are essential to

chromosome segregation during mitosis, microtubule removal later in mitosis is required for normal nuclear morphology in the subsequent interphase.
In Xenopus egg extracts, chromatin-bound Dppa2 was shown to be required


New Insights into Mechanisms and Functions of Nuclear Size Regulation

15

to inhibit microtubule polymerization around the chromosomes during NE
assembly, and persistent microtubules led to the formation of small, misshapen nuclei (Xue and Funabiki, 2014; Xue et al., 2013). Also see Sections
3.2, 3.4, 3.7[48_TD$IF], and 4.1.

2.9 Signaling Pathways
While manipulating the levels or activities of NE components can alter the
size and shape of the nucleus, relatively few studies address mechanisms of
nuclear size regulation in a physiological context. The Xenopus embryo
provides an excellent model system for studying nuclear size regulation.
Upon fertilization, the single-cell Xenopus embryo undergoes a series of
rapid divisions to generate a few thousand cells, while the overall size of
the embryo remains unchanged. Dramatic reductions in cell size during early
development are associated with changes in nuclear size and dynamics,
without changes in nuclear DNA content (Jevtic and Levy, 2015; Levy
and Heald, 2010) (Fig[21_TD$IF]. 1). The Xenopus egg and embryo extract systems
constitute undiluted cytoplasms that have been extensively used to study
various cellular activities including nuclear assembly and import, mitotic
spindle regulation, and chromosome structure (Chan and Forbes, 2006;
Edens and Levy, 2014b; Good et al., 2013; Hara and Merten, 2015; Hazel
et al., 2013; Kieserman and Heald, 2011; Levy and Heald, 2010; Loughlin
et al., 2011; Wilbur and Heald, 2013).
We sought to develop nuclear re-sizing assays, using Xenopus embryo

extracts, in order to identify novel regulators of nuclear size. We found that
large nuclei, assembled in Xenopus egg extract, became smaller when incubated in cytoplasm isolated from late stage embryos. Using this system, we
determined that this nuclear shrinking activity was regulated by the activation and nuclear translocation of conventional protein kinase C (cPKC),
leading to removal of lamins from the NE. During development, nuclear
cPKC activation and localization increase, correlating with decreased
nuclear size (Fig[21_TD$IF]. 1). Furthermore, we showed that this signaling pathway
was also important for proper nuclear size regulation in vivo in the embryo
during interphase (Edens and Levy, 2014a). While PKC activity has previously been implicated in NEBD during mitosis and nuclear export of large
ribonucleoprotein complexes and certain viruses (Hatch and Hetzer, 2014;
Leach and Roller, 2010; Milbradt et al., 2010; Park and Baines, 2006; Speese
et al., 2012), our data suggest that interphase nuclear cPKC activity plays a
role in steady-state nuclear size regulation (Fig[21_TD$IF]. 3).


16

[(Figure_3)TD$IG]

Lidija D. Vuković et al.

(A)

Pre-MBT:
Nuclear growth

(B)
MBT &
post-MBT:
Nuclear
shrinking

(C)
Equilibrium
balance
Model:
Steady-state
nuclear size

Phospholipid double bilayer

Active conventional protein kinase C

Lamin protein dimers, tetramers etc.
associated in meshwork

Inactive conventional protein kinase C

Phosphorylated, dissociated lamin protein

Nuclear pore complex

Chromatin

Importin α

Figure 3 [1_TD$IF]Models of nuclear size regulation during Xenopus development. [12_TD$IF](A) In the preMBT embryo, nuclear growth is mediated by greater importin α activity and import of
lamin B3. (B) In the post-MBT embryo, nuclear shrinking is mediated by increased
nuclear cPKC localization and activity, and subsequent dissociation of lamins from the
NE. (C) A balance of import and cPKC-mediated shrinking determines steady-state
nuclear size. This model is based on our studies of nuclear size regulation in Xenopus

(Edens and Levy, 2014a; Levy and Heald, 2010).


New Insights into Mechanisms and Functions of Nuclear Size Regulation

17

While nuclear lamins are known targets for PKC phosphorylation
(Simon and Wilson, 2013), it remains to be determined whether nuclear
shrinking is mediated by direct PKC phosphorylation of lamins or intermediate signaling proteins. Consistent with our results, the phosphorylation
state of lamin A in HeLa cells mediates its assembly dynamics with the lamina
meshwork during interphase (Kochin et al., 2014). To account for the
mechanical forces associated with lamin removal during nuclear shrinking,
we envision a model wherein lamin density must remain roughly constant for
the structure of the nucleus to maintain its integrity and withstand cytoskeletal forces (Buxboim et al., 2010). By this model, increased lamina dynamics
and loss of lamins from the NE are compensated by a retraction of NE
membrane back into the ER, thus maintaining a constant nuclear lamin
density as nuclear size becomes smaller.
While diverse models of organelle size regulation have been discussed
elsewhere (Chan and Marshall, 2012; Goehring and Hyman, 2012; Webster
et al., 2009), we envision two plausible mechanisms for how nuclear size is
determined during early Xenopus development. The first model posits that
there is a limiting component for nuclear growth and as this component is
distributed among greater numbers of cells during development, nuclei
reach a steady-state size that scales smaller with cell number. Possible limiting
components include molecules that directly inhibit cPKC or an inhibitor of
an upstream cPKC activator. One might also consider the affinity of PKC for
its various substrates, which can vary drastically and is important in differential spatial and temporal regulation of PKC activity (Fujise et al., 1994).
Over the course of development, changes in the relative abundances of
different PKC substrates with varying affinities might shift PKC activity

toward lower affinity substrates relevant to nuclear shrinking.
A second model is based on the idea that there is an equilibrium balance
between nuclear growth and contraction. Such equilibrium balance models
have been applied to the mitotic spindle (Loughlin et al., 2011), flagella
(Marshall et al., 2005), mitochondria (Rafelski et al., 2012), vacuoles
(Chan and Marshall, 2014), nucleus (Edens and Levy, 2014a), and others
(Chan and Marshall, 2010, 2012). In the case of a membrane-bound organelle like the nucleus, the simplest equilibrium balance model is one where a
constant rate of nuclear growth, mediated by nuclear import, is balanced by a
proportional contraction rate mediated by cPKC translocation to the NE. By
balancing nuclear import-mediated growth, nuclear shrinking causes nuclei
to reach a steady-state size (Fig[21_TD$IF]. 3). It is important to note that the equilibrium balance model is not mutually exclusive with limiting component


18

Lidija D. Vuković et al.

models of size regulation (Goehring and Hyman, 2012). Important questions
remain about what upstream regulatory signals might control nuclear size in
this system. Although several factors that influence nuclear size are known,
an integrated model of the regulatory mechanisms controlling nuclear size
has yet to be described.

3. MODEL SYSTEMS TO ELUCIDATE MECHANISMS
OF NUCLEAR SIZE REGULATION
Cells tend to maintain their nuclear size within a defined range.
Changes in cell size that occur during development, cell division, and
differentiation are accompanied by dynamic nuclear size adjustments in
order to establish appropriate N/C volume relationships. Mechanisms that
regulate proper nuclear size and the functional significance of this regulation

are largely unknown. Aberrations in nuclear size are associated with certain
disease states, most notably cancer. It seems likely that nuclear size and the N/
C volume ratio affect cell physiology, for instance through altered chromatin
organization and gene expression. In this section we focus our attention on
studies from different eukaryotic model experimental systems including
Tetrahymena, yeasts and fungi, plants, worms, flies, fish, frogs, mice, and
mammalian tissue culture. Research based on these model systems has elucidated some important molecular mechanisms of nuclear size regulation
(Fig[21_TD$IF]. 2).

3.1 Tetrahymena thermophila
The ciliateTetrahymenathermophila has two morphologically and functionally
distinct nuclei both located within the same cell. The bigger somatic macronucleus (MAC) is polyploid and transcriptionally active, while the smaller
germinal micronucleus (MIC) is diploid and transcriptionally inactive during
the vegetative growth cycle (Frankel, 2000). The two nuclei also differ in
protein composition. Macronuclear linker histone H1 is localized to the
MAC and its deletion results in enlargement of only the MAC.
Conversely, micronuclear linker histone (MLH) is unique to the MIC and
its deletion leads to enlargement of the MIC but not MAC (Allis et al., [49_TD$IF]1979,
1980; Shen et al., 1995; White et al., 1989).
Among all identified nucleoporins, four homologues of Nup98 were
found to have strict nuclear selectivity, with two localized to the MAC


New Insights into Mechanisms and Functions of Nuclear Size Regulation

19

(MacNup98A and MacNup98B) and the other two localized to the MIC
(MicNup98A and MicNup98B). MacNup98A and MacNup98B have typical GLFG N-terminal repeat domains, while MicNup98A and MicNup98B
have unusual repeats of NIFN or SIFN. To elucidate the function of MACspecific and MIC-specific repeats, swapping experiments of N-terminal

repeat domains between MacNup98A and MicNup98A were performed.
Chimeric proteins composed of the N-terminal NIFN repeat domain of
MicNup98A and the C-terminal domain of MacNup98A (BigMac) exclusively located to the MAC NPC, and chimeric proteins composed of the Nterminal GLFG repeat domain of MacNup98A and the C-terminal domain
of MicNup98A (BigMic) predominantly located to the MIC NPC.
Overexpression of BigMic caused a 2-fold increase in the size of MIC and
a dramatic decrease in MIC localization of MIC-specific MLH proteins.
BigMac overexpression led to increased MAC size and decreased MAC
localization of macronuclear histone H1 (Iwamoto et al., 2009). These data
demonstrate that, to enable nucleus-selective import of different proteins,
the MAC and MIC utilize unique Nups.
Karyopherins also contribute to the regulation of nuclear size. In total,
Tetrahymena encodes 13 putative importin α-like proteins and 11 importin
β-like proteins. Nine importin α proteins are MIC specific, while all importin β proteins localize to both the MIC and MAC. Knockdown of IMA10, a
MIC-specific importin α, caused MIC division defects including lagging
MIC chromosomes, loss of MIC DNA content, and abnormal nuclear
number and morphology. This suggests that IMA10 plays a MIC-specific
role in regulating MIC division and nuclear morphology. Transport to the
MIC and MAC are mediated through different subsets of importin α transport receptors that are uniquely targeted to each nucleus, and this likely has
important implications for how nuclear size is regulated in the two types of
nuclei, for instance through regulated import of different histone H1 isoforms (Malone et al., 2008).
Treatment ofTetrahymena with low concentrations of the DNA polymerase
α inhibitor aphidicolin led to cell division arrest and, surprisingly, rounds of
MAC endoreduplication and cell size increase. Upon resumption of cell
division, large extrusion bodies formed from dividing MACs and the size of
extrusion bodies correlated with the duration of aphidicolin pre-treatment
and the amount of MAC DNA (Kaczanowski and Kiersnowska, 2011). These
data suggest that compensatory mechanisms exist to regulate the level of ploidy
inTetrahymena, thereby influencing nuclear morphology and cell size.